Abstract:

A method for separating and purifying the active hematinic species (AHS)
present in iron-saccharidic compositions, including AHS such as sodium
ferric gluconate complex, ferric hydroxide-sucrose complex and ferric
saccharate complex and others of similar form and function. The method
separates the AHS from one or more excipients and, preferably,
lyophilizes the separated AHS. Separation of the AHS permits its
analytical quantification, further concentration, purification and/or
lyophilization as well as preparation of new and useful products and
pharmaceutical compositions, including those useful for the treatment of
humans and animals.

Claims:

1. A composition comprising an active hematinic species (AHS) for treating
an animal, wherein said composition is substantially free of excipients
having a molecular weight of less than about 5,000 Daltons.

3. The composition of claim 1 wherein said excipients are present at less
than about 5 weight percent.

4. The composition of claim 1 further comprising a solvent or diluent for
said AHS or a mixture of solvent and diluent.

5. The composition of claim 4 wherein said solvent or diluent comprises
water.

6. The composition of claim 1, said AHS having an absolute molecular
weight of at least about 100,000 Daltons.

7. The composition of claim 6 further comprising a solvent or diluent or a
mixture of solvent and diluent in admixture therewith, said mixture
suitable for parenteral administration.

8. The composition of claim 7 wherein said solvent or diluent comprises
water.

9. The composition of claim 1 wherein said excipients include byproducts
and unreacted starting materials from the synthesis of said AHS,
degradation byproducts, iron containing species having a molecular weight
of less than about 5,000 Daltons and non-hematinically active components.

10. The composition of claim 9 wherein said non-hematinically active
components include sucrose.

11. The aqueous composition of claim 8 wherein said AHS comprises ferric
iron present at a concentration of 50-120 mg per 5.0 milliliters volume
of said composition.

12. The composition of claim 2 comprising greater than about 85 weight
percent of AHS and, correspondingly, less than about 15 weight percent
excipients having a molecular weight of less that about 5,000 Daltons.

13. The composition of claim 12 wherein said excipients are present at
less than about 2 weight percent.

15. The composition of claim 1 wherein: (a) said AHS is in powder form and
comprises about 30% to about 95% by weight of said composition; (b) said
composition further comprises a parenterally acceptable buffering agent
in an amount of about 5% to about 60% by weight; and (c) said composition
optionally comprises one or more parenterally acceptable excipient
ingredients in a total amount of zero to about 10% by weight, of the
composition; said composition being reconstitutable in a parenterally
acceptable liquid.

22. The composition of claim 15 in admixture with an aqueous carrier or
solvent.

23. The composition of claim 22 wherein the aqueous carrier or solvent
further comprises sodium chloride, a preservative or both sodium chloride
and a preservative.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001]This application is a continuation of U.S. application Ser. No.
11/176,132 filed on Jul. 7, 2005, which is a division of U.S. application
Ser. No. 10/600,173 filed on Jun. 20, 2003, which is a
continuation-in-part of U.S. application Ser. No. 10/371,783 filed on
Feb. 21, 2003, now U.S. Pat. No. 6,773,924, issued on Aug. 10, 2004,
which is a division of U.S. application Ser. No. 09/999,394 filed on Oct.
31, 2001, now U.S. Pat. No. 6,537,820, issued on Mar. 25, 2003, further
claiming the benefit of U.S. Provisional Patent Application No.
60/245,269, filed Nov. 2, 2000, the disclosures of which are hereby
incorporated by reference herein.

BACKGROUND OF THE INVENTION

[0002]The present invention relates to therapeutically active
iron-containing species including parenteral hematinic pharmaceuticals.
For purposes of the present invention a "hematinic" means a compound or
composition comprising iron in a form that tends to increase the amount
of hemoglobin in the blood of a mammal, particularly in a human. While
such compounds can be broadly characterized as iron-carbohydrate
complexes, which can include dextrans, the present invention is directed
to the generic subclass known as iron-saccharidic complexes and includes
such species as sodium ferric gluconate complex in sucrose (SFGCS),
ferric hydroxide-sucrose complex (FHSC) and/or others characterized as
iron saccharates. For purposes of the present invention, such active
iron-containing species are referred to generically as iron-saccharidic
complexes or active hematinic species (AHS). The term "complex" may have
alternate meanings in various contexts in the related art. In one aspect,
the term complex may be used to describe the association between two or
more ions to form a relatively low molecular weight non-polymeric
composition which exists singly under a given set of conditions. This
type of complex has been referred to as a "primary complex". An alternate
manner in which this term is used is to describe an association or
agglomeration of a plurality of primary complexes into a large
macromolecule, or "secondary complex." For purposes of the present
invention, the latter agglomerates are also referred to herein as
macromolecules. For the purposes of the present invention, such
macromolecules or secondary complexes are identified as "complexes" and
are referred to simply as complexes. As an example of the above
distinction, ferrous gluconate is a composition comprising divalent iron
ions and gluconate anions. A divalent iron ion and two gluconate anions
form a primary complex of relatively low molecular weight (about 450
Daltons) and primary complexes of this type do not become agglomerated
into macromolecules when dissolved into an aqueous medium. Ferrous
gluconate, therefore, is a not composition which falls within the scope
of the term "complex" herein. Ferric gluconate, however, does exist as a
complex as that term is used herein because primary complexes of
trivalent iron ions and gluconate anions agglomerate to form large
macromolecules (and can have molecular weights of from about 100,000 to
about 600,000 Daltons, or more). Several embodiments of therapeutically
active ferric iron compounds are commercially available, as will be
described below. For purposes of the present invention, the term
"excipients" means non-hematinically active components, including
synthesis reaction by-products and unreacted starting materials,
degradation by-products, diluents, etc., present in admixture with
therapeutically active iron-containing species such as iron-saccharidic
complexes. Such excipients can include one of more sugar, such as
sucrose, that may be present in combination with the AHS following
synthesis, as an unreacted or partially reacted component, or added to
the AHS in the course of preparing a parenteral composition, e.g.,
commercially available parenteral iron compositions as described below.

[0003]Iron deficiency anemia is a blood disorder that can be treated using
various therapeutic preparations containing iron. These preparations
include simple iron salts such as ferrous sulfate, ferrous gluconate,
ferrous fumarate, ferrous orotate and others. Various low molecular
weight iron, Fe(III), compounds intended for use as oral or nutritional
supplements are known. Such low molecular weight compounds are only
useful as oral supplements, since the introduction of materials having
high unit concentrations of iron directly into the bloodstream by
injection would be contraindicated and could be toxic. In contrast, the
compounds of the present invention, intended for parenteral use, have
lower iron concentrations and can be used parenterally. For purposes of
the present invention, parenteral means introduced into the body by some
other means than through the gastrointestinal tract; for example, by
intradermal, subcutaneous, intramuscular, intravenous, intramedullary,
intra-articular, intrasynovial, intraspinal, intrathecal or intracardiac
injection or infusion.

[0004]If the use of such orally administered substances fails to
ameliorate iron deficiency, the next level of treatment includes
parenteral iron administration. Depending on a patient's clinical status,
parenteral administration of polyglucan or dextran-linked iron may serve
as an effective therapeutic iron-delivery vehicle. Intramuscular
injection or intravenous routes may be used to administer these iron
dextrans; commercial examples of such products include those having trade
names such as "Imferon", and "INFeD". Various clinical conditions that
require parenteral iron have shown the practical hematinic value of iron
dextrans. The use of iron dextrans is tempered by idiosyncrasies in their
synthesis, manufacturing and patient responses such as hypersensitivity.
These effects may be exhibited as a severe allergic response evident as
anaphylaxis or symptoms as minor as transient itching sensations. Whether
such allergic or other adverse effects are due to individual patient
sensitivity to the active ingredient or to byproducts, impurities or
degradation products in the parenteral solution has not been established.

[0005]As an alternative to iron dextrans, iron-saccharidic complexes are
regarded herein as non-dextran hematinics. Whereas the iron dextrans
comprise polymerized monsaccharidic residues, the iron-saccharidic
complexes of the present invention are characterized by the substantial
absence of such polymerized monosaccharides. Iron-saccharidic complexes
are commercially available, for example, under the tradename Ferrlecit,
which is identified as sodium ferric gluconate complex in sucrose
(SFGCS). The manufacturer states that the structural formula of the
product is considered to be
[NaFe2O3(C6H11O7)(C12H22O11)5]n, where n is about 200, and as having an apparent molecular weight
of 350,000±23,000 Daltons. However, it is noted that, based on the
published structural formula just recited, the formula weight should be
significantly higher, 417,600 (although, as published, the formula is
difficult to accurately interpret). Furthermore, the commercial hematinic
composition comprises 20% sucrose, wt./vol. (195 mg/mL) in water. The
chemical name suggests that therapeutic iron (Fe) in this form is
pharmacologically administered as the oxidized ferric form Fe(III) as
opposed to the reduced ferrous Fe(II) form. Owing to the charged
oxidation state of Fe(III) it has been suggested that gluconic acid
(pentahydroxycaproic acid, C6H12O7) also exists in a
coordination complex or ligand form in a sucrose solution. For purposes
of the present invention it is to be understood that the chemistry of
gluconate, whether held in a ligand complex with Fe(III) or not, does not
exempt it from interactions with other carbohydrates that may be present,
such as sucrose. Thus, use of the term iron-saccharidic complex will be
understood to indicate the existence of a nonspecific and imprecise
structure where ionized gluconic acid (gluconate) and sucrose molecules
are tenuously associated by various bonding interactions to give a
molecular scaffolding that incorporates Fe(III). Another non-dextran
hematinic of the present invention is compositionally described as ferric
hydroxide-sucrose complex (FHSC). This parenteral hematinic is
commercially available under the tradename "Venofer". As with SFGCS, the
descriptive name suggests a form of ferric iron, Fe(III), that is present
in a spatial complex with sucrose or some derivative of sucrose.
Therefore, non-dextran, iron-saccharidic complexes of the present
invention include SFGCS, FHSC and mixtures thereof. These iron delivery
vehicles include an iron-containing structural complex that, for purposes
of the present invention, is designated the active hematinic species
(AHS).

[0006]For purposes of the present invention, the term AHS is used
interchangeably with iron-saccharidic complex, saccharidic iron delivery
vehicle, and iron saccharate. The term "saccharate" or "saccharidic" is
employed to generically describe iron atom interactions with another
individual molecule or its polymers that display a saccharose group
structurally identified as

--CH(OH)--C(O)--

[0007]The simplest occurrence of the saccharose group is where the two
terminal positions in a standard Fischer molecular projection model of a
molecule appear as an ald- or a keto-group respectively designated as:

(--CH(OH)--CHO) or (--CHO--CH2OH).

[0008]This nomenclature format is also described in Zapsalis, C. and R. A.
Beck, 1985, "Food Chemistry and Nutritional Biochemistry," Chapter 6,
John Wiley & Sons, pp. 315-321 (incorporated herein by reference to the
extent permitted). Such groups and their first oxidation or reduction
products occur in molecules recognized as monosaccharides that contain
carbon atoms with hydrogen and oxygen in the same ratio as that found in
water. By way of example, the aldose sugar known as glucose would have
gluconic acid as a first oxidation product and glucitol, also known as
sorbitol, as a first reduction product. Both the original monosaccharide
represented by the model of glucose and its possible reaction products
retain evidence of the characteristic saccharide group in an oxidized or
reduced form. While these structural variations exist, both remain
recognized as monosaccharides and carbohydrates. In practical
nomenclature, the oxidized version of the saccharose group exhibits a
carboxyl group which under the appropriate pH conditions will allow it to
ionize according to its unique ionization constant and pKa value.
When ionized, the oxidized saccharose group is denoted as a "saccharate"
or it can be generically described as a saccharidic acid where the
ionizable proton remains with the oxidized saccharose group. If the
ionized carboxyl group of the saccharose group is associated with a
cation such as sodium, a saccharidic acid salt is formed. For example,
oxidation of glucose gives gluconic acid and the sodium salt of this
saccharidic acid is sodium gluconate. Similarly, where a ferrous (FeII)
cation is electrostatically associated with the carboxyl group of
gluconic acid, ferrous gluconate results. Monosaccharides that are
aldoses commonly undergo oxidation to give their saccharidic acid
equivalents or, when ionized, monosaccharate forms may interact with
selected cations having valence states of +1 to +3. Glyceraldehyde is the
simplest structure that demonstrates such an ald-group while
dihydroxyacetone serves as a corresponding example of a keto-group.
Practical extensions of such structures with six carbon atoms account for
the descriptive basis of two carbohydrate classifications, one form being
aldoses and the other ketoses. Aldoses and ketoses are respectively
represented by monosaccharides such as glucose or fructose. With many
possible intra- and intermolecular reaction products originating from
monosaccharides, including the glucose oxidation product known as
gluconic acid, efforts to complex iron with saccharates can produce an
AHS. For purposes of the present invention, AHS is considered to be a
more chemically complex embodiment of hematinic iron than suggested by
the generic descriptor sodium ferric gluconate complex in sucrose (SFGCS)
or ferric hydroxide-sucrose complex (FHSC), and therefore, designations
including iron-saccharidic complex or saccharidic-iron delivery vehicle
or saccharidic-iron are used interchangeably with AHS. Consequently,
intra- and inter-molecular reactions or associations from reactions of
monosaccharides with iron during hematinic syntheses can coincidentally
produce a wide variety of structural species with hematinic properties
that are encompassed within the present invention.

[0009]Typically iron-dextrans are provided for delivery of up to 100 mg
Fe(III)/2.0 milliliter (mL) of injectable fluid, whereas iron-saccharidic
complexes can provide 50-120 mg of Fe(III)/5.0 mL volume as commercially
prepared in a single dose. As made, many of these iron-saccharidic
complex products contain 10-40% weight-to-volume occurrences of
non-hematinic excipients as well as synthesis reaction by-products.

[0010]While some hematinic agents have an established compendial status
under the aegis of the United States Pharmacopoeia (USP) or National
Formulary (NF), iron-saccharidic complexes have no acknowledged
compendial reference, standardized molecular identity characteristics or
documented molecular specificity unique to the active hematinic species.
This suggests that the iron-delivery vehicle in non-dextran hematinics
such as SFGCS or FHSC has not previously been adequately purified and
separated from manufacturing excipients so as to permit detailed
characterization. Consequently, there has not been developed a benchmark
reference standard or an excipient-free analytical quality control index
capable of characterizing one desirable hematinic agent from others
having uncertain characteristics. Since the 1975 merger of the USP with
the NF to produce the USP-NF compendial guidelines for drugs, standard
identities and analytical protocols have been developed for over 3,800
pharmaceuticals while 35% of marketed pharmaceuticals are still not
included in the USP-NF. Hematinic pharmaceuticals such as SFGCS and FHSC
fall within this latter category. This issue has been recently addressed
in "Raising the Bar for Quality Drugs", pp. 26-31, Chemical and
Engineering News, American Chemical Society, Mar. 19, 2001. As in the
case of immune and anaphylactic responses elicited by specific antigens,
a fine line of molecular specificity and compositional differentiation
can separate a no-adverse-effect level for one hematinic's active
molecular structure and excipients from another that may induce such
adverse reactions. Thus, there is a need to identify features that
document one hematinic's safe and effective characteristics from others
where little is known about the iron-delivery vehicle, excipients
representing synthesis reagent overage or byproducts of hematinic
synthesis reactions. Furthermore, there are no long-term detailed sample
archives or data using modern analytical instrumentation that
meaningfully characterize the chemical nature of even the safest
parenteral iron-saccharidic complexes. Moreover, correlation between
variations in normal hematinic manufacturing conditions and their
consequential effects identifiable as changes in the chemical structure
of a released pharmacological agent have not been identified. The methods
of the present invention can address such issues.

[0011]The present invention can also provide an analytical basis for a
routine protocol in order to fingerprint and characterize
iron-saccharidic complexes such as SFGCS, FHSC and others as well as
discriminate between competing products and structural transformations
exhibited by an individual product.

[0012]The need to characterize an AHS is also reflected in the quality
control demands of manufacturing processes, particularly where
endothermic conditions and heat transfer issues can affect final product
quality. Whatever the proprietary synthesis process, possible heat-driven
or Strecker reaction byproducts in some commercially released non-dextran
products suggest that hematinic product formation is contingent on at
least some controlled heat-input during the course of manufacturing. Such
excipients would not occur if process temperatures less than about
50° C. were unnecessary. It follows then, that product quality is
related, to some extent, to issues of heat transfer rates and duration of
heat exposure. Where products are especially sensitive to heat processing
conditions, knowledge of excipient profiles can also provide significant
insight to the product quality of the active pharmacological substance.
In other words, monitoring the safe and effective pharmacological agent
can also be indicated by the nature and constancy of excipient occurrence
in a drug as released into the marketplace.

[0013]Analytical studies on iron-saccharidic complexes, including AHS and
its coexisting excipients are hampered by factors of low concentration,
molecular interactions, over-lapping analytical signals and so on. For
both SFGCS and FHSC, analytical challenges include high concentrations of
hydrophilic excipients, including excess reactants and reaction and
post-reaction byproducts, from which their respective AHS's have not
previously been isolated or reported in terms of their individual
properties. Reference standards for pharmaceuticals need to abide by
practical protocols that are routinely achievable using methods that are
analytically discriminating and able to be verified and validated. There
is a continuing need for such methods and application of the present
invention can facilitate compliance with such protocols as well as
verifying manufacturing consistency and product stability.

SUMMARY OF THE INVENTION

[0014]A method for measuring at least one molecular characteristic of an
iron-saccharidic complex, the complex comprising at least one active
hematinic species (AHS) and one or more excipients, the AHS having a
determined value of differential refractive index increment (dn/dc), the
method comprising: (A) subjecting the complex to liquid chromatographic
analysis (LCA) having a refractive index (RI) detector and in-line eluate
stream detection using laser light scattering (LLS); and (B) calculating
the molecular characteristics based on the dn/dc value. In a particularly
preferred method the dn/dc value is determined by (a) substantially
separating the AHS from the one or more excipients to obtain purified
AHS; and (b) measuring the dn/dc value of the purified AHS. The method is
particularly useful for calculating molecular characteristics such as
absolute molecular weight, molecular weight distribution, size, shape,
morphology, and dimensional variations.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIG. 1 shows the chromatographic signature obtained for isolated and
purified AHS or primary reference standard of an iron-saccharidic complex
as isolated in Fraction 1.

[0018]FIG. 4 shows LLS and RI data based on HPLC analysis of a commercial
sample of iron-saccharidic complex and indicating structural deviations
from AHS or primary reference standard indicated as active hematinic
species aggregate peak (AHSAP)

[0019]FIG. 5 shows LLS and RI based on HPLC analysis of a second
commercial sample of iron-saccharidic complex and indicating structural
deviations from AHS or primary reference standard indicated as active
hematinic species aggregate peak (AHSAP)

[0020]FIG. 6 shows LLS and RI data based on HPLC analysis of a sample of
iron-saccharidic complex and indicating an iron aggregate peak
(AHSAP.sup.TAM2) by time interval 1 after its manufacture.

[0021]FIG. 7 shows LLS and RI data based on HPLC analysis of a sample of
iron-saccharidic complex and indicating an iron aggregate peak
(AHSAPTAM2) by time interval 2 after its manufacture.

[0022]FIG. 8 shows LLS and RI data based on HPLC analysis of a sample of
iron-saccharidic complex and indicating an iron aggregate peak
(AHSAPTAM3) by time interval 3 after its manufacture.

[0023]FIG. 9 shows LLS and RI data based on HPLC analysis of a sample of
iron-saccharidic complex and indicating an iron aggregate peak
(AHSAPTAM4) by time interval 4 after its manufacture.

[0024]FIG. 10 shows the chromatographic signature for an iron-saccharidic
complex isolated as Fraction 1, lyophilized, reconstituted and analyzed
using RI and LLS-based HPLC.

DETAILED DESCRIPTION

[0025]Prior to the present disclosure, the AHS responsible for parenteral
iron-delivery had not been suitably separated from its excipients with
controlled, reproducible purity. Such co-mingling of excipients with the
AHS challenged the development of improved hematinic products as well as
characterization of the AHS. In view of the improved methods disclosed
herein, opportunities are afforded for analytical monitoring of the
iron-saccharidic complex to gauge product compliance with manufacturing
specifications, to determine storage stability indices and batch-to-batch
comparison using a benchmark reference standard. Such a benchmark
reference standard has not previously been available, particularly since
the AHS had not been isolated. The methods of the present invention allow
therapeutically active AHS to be effectively separated and concentrated
from coexisting excipients, and then to be dried, or lyophilized, if
desired. The present invention provides the information, methods and
analytical details for the preparation, characterization and use of
sodium ferric gluconate complex in sucrose, SFGCS, ferric hydroxide
sucrose complex, FHSC and iron saccharates. The materials are generally
referred to as iron-saccharidic complexes or hematinics. It also provides
methods for establishing reference standards for these materials that
were heretofore non-existent. The disclosure also provides the analytical
basis for routine discrimination in connection with manufacturing and
continued monitoring of these products after release. The analytical and
other methods disclosed herein are generally applicable to
iron-saccharidic complexes including commercial and generic
iron-saccharidic complexes as well as current commercial parenteral
iron-saccharidic complexes that perform a hematinic function.

[0026]Prior to development of the methods disclosed herein, standards for
iron-saccharidic complexes were not available because there was no method
to separate, purify and characterize the AHS without unduly affecting or
destroying the AHS. However, nondestructive characterization of the AHS
using high pressure liquid chromatography in combination with laser light
scattering combine to provide an analytical method for safely and
accurately characterizing such saccharidic-iron complexes. Coupled with
the separation and purification process taught herein, there is now a
method for providing AHS reference standards with the further opportunity
to use such materials in specifying products.

[0027]According to the present invention, a method for characterizing the
AHS in the absence of its excipients includes separating the AHS and
associated excipients into at least two fractions. Since the AHS
ordinarily exists at very low concentrations, once separated, it is also
preferable to concentrate the AHS in order to permit its detailed
analytical study. Although AHS are subject to degradation, they can be
concentrated, provided that hydrophilic excipients are substantially
absent from the composition prior to or at the time of concentration.
Concentration can be effected by drying and, preferably, by
lyophilization, but other methods can be employed. After drying,
preferably by lyophilization, the AHS is in powder form.

[0028]Weak electron bonding interactions such as hydrogen bonding between
water and excipients provide challenges in the separation and drying of
the AHS. As released, for example in parenteral form, the AHS typically
is present in an aqueous system that has a lower water activity (Aw)
than solute-free water, i.e., pure, water. It is generally accepted that
solute-free water has an Aw value approaching or equal to 1.0. Water
activity is a parameter corresponding to the availability of the water
present in a substance or composition for participating in physical,
chemical and biological processes. Water activity is reduced as a
consequence of the presence of water soluble or hydrophilic substances,
in particular those having a low molecular weight. In other words, a
portion of the water present corresponding to the fraction of the water
which serves to bond or dissolve the substances is otherwise unavailable.
Consequently, water activity diminishes as the number and/or
concentration of substances dissolved or bound increases. As most often
applied in the food and pharmaceutical industries, elevated water
activity values are associated with chemical and biological degradation
so that water soluble or hydrophilic substances are added in order to
decrease the value of Aw. In the present invention, solutes and/or
colloids, including materials suspended or dispersed in the aqueous
composition, including saccharidic excipients accompanying the AHS,
interact with water, e.g., they are hydrophilic or they can hydrogen
bond. Such substances reduce the Aw value for water present to a
value less than 1.0. So long as hydrophilic excipients are present in
water in which the AHS is present, a portion of the water remains bound
to such excipients and removal of water from the composition comprising
the AHS, accompanied by its concentration, cannot acceptably be achieved.
Consequently, separation of hydrophilic excipients from the AHS increases
the Aw value toward 1.0 in the phase or fraction comprising the AHS,
and facilitates the concentration, drying and analysis of these preferred
species.

[0029]Solute effects on water in systems comprising SFGCS and FHSC
directly impact upon the drying behavior of these species, including
lyophilization, because lowering of their released hydrophilic and
excipient concentrations is accompanied by an increased water vapor
pressure and an associated increase in the Aw. The concept of water
activity, expressed as Aw, has found its most significant use in the
area of food science (see, for example, "Principles of Food Science",
edited by O. R. Fennema, "Part II, Physical Principles of Food
Preservation", M. Karel, et al., pp. 237-263, Marcel Dekker, Inc. 1975;
Enclyclopedia of Food Science, edited by M. S. Peterson, et al., "Water
Activity in Relation to Food", D. H. Chou, pp. 852-857, Avi Publ. Co.,
Inc., 1978, each incorporated herein to the extent permitted). The
premise is that there is a relationship between the chemical and physical
processes that may occur in food storage and the amount and state of
water in the foods. The same principles can be applied to other
substances or compositions in which water is present and which can be
affected by water. Water present in a composition, as a solvent, diluent
or otherwise, can be characterized as free or unbound or bound to various
degrees to other compounds, for example, in the present case to
excipients or the AHS. Various methods can be used to determine the
degree to which the water is bound, including determining the amount of
unfrozen water in a water containing composition below 0° C.,
measurement by nuclear magnetic resonance, determination of dielectric
properties and measurement of vapor pressure; the latter technique is
preferred for its simplicity. In this method, water activity is defined
as the ratio of partial pressure of water in the composition or compound
in which water is present, to the vapor pressure of pure water at a given
temperature. This follows from Raoult's Law where water vapor pressure
present in a solution (P) is compared to the vapor pressure of pure water
(solute-free,) (Po). As the ratio P/Po increasingly drops below
1.0, the Aw value becomes smaller, the entropy state for such water
decreases, its vapor pressure is lowered, presumably as a consequence of
increased water-solute binding interactions, and vapor phase removal of
water becomes more difficult. These conditions influence drying processes
such as lyophilization. Furthermore, in complex mixtures, in other words,
where chemical compounds or complexes may not be completely dissolved or
present in the form of a true solution, there can be substantial
deviations from the ideal Raoult's Law expression and, furthermore, water
activity of the components comprising the composition can differ. It has
also been observed in multiphasic compositions that transport or
diffusion of water from a component in which Aw is higher to one where it
is lower, can occur contrary to what might otherwise be expected based on
concentration driving forces (M. Karel et al., page 251). It should also
be appreciated that, as a complex composition is dried, the concentration
of solutes having differing degrees of interaction with water, for
example, different levels of hydrogen bonding, can affect, in a complex
and changing way, the ability to remove the water. This is a further
important reason to substantially separate or isolate the active
hematinic species from their excipients so that appropriate drying
process conditions can be determined and controlled. Water can be
strongly bound to specific sites on compounds, including the hydroxyl
groups of polysaccharides, as well as to carbonyl and amino groups and
others on which water can be held by hydrogen bonding, by ion-dipole
bonds, or by other strong interactions. Therefore, in the present case,
water can be bound to specific sites on the AHS and excipients, for
example, as a monolayer. Such water can be present as non-freezable
water, unless temperatures are significantly below 0° C.;
additionally, it can be present as non-solvent water. In the present
compositions, water is known to bind strongly to sucrose so that Aw
in a sucrose-containing composition is depressed. Therefore, drying,
particularly freeze drying (described in detail below), can be adversely
affected.

[0030]Whether lyophilized (freeze-dried) or not, the ability to prepare
purified AHS as a discrete substance provides significant capabilities
for defining the chemical and structural features that characterize such
hematinic products. With verifiable and reproducible parameters
established for such a hematinic, it is possible to establish a quality
control framework of, for example, batch-to-batch uniformity of the AHS.
Departures from validated structural uniformity in the AHS can be used as
an index of storage stability and to assist in identifying cause-effect
relationships of variables in manufacturing processes. The ability to
isolate and analytically characterize these agents using modern
technology, particularly laser light scattering, helps to define the
attributes of such iron-saccharidic complexes.

[0031]Little is known about the synthesis of iron-saccharidic complexes
for hematinic use because such syntheses have generally relied on
proprietary methods. Similarly, there is little analytical information
available for such complexes. The active pharmacological hematinic
species or iron-delivery vehicle in the hematinic is believed to be less
than five percent according to weight-by-volume measurements of total
solids and it may be as low as 1.0 percent. Thus, in commercial products,
measurement of the characteristics of the AHS as released has typically
been carried out in the presence of a high percentage of hydrophilic
excipients. Such low concentrations of the unique ferric iron-delivery
vehicle accompanied by relatively large amounts of excipients challenged
characterization of the AHS. Furthermore, no reference standard had been
established for AHS since it was believed to be so unstable that it could
not be isolated for analysis. With the effective separation of AHS from
its excipients based on the methods described herein, these methods can
be extended so as to develop improved hematinic agents alone or admixed
with other pharmacological agents. For example, purified AHS can be
combined with erythropoietin or other useful drugs.

[0032]Access to excipient-free AHS permits closer analytical monitoring of
the AHS and its pharmaceutical performance. Moreover, for pharmaceuticals
where excipients account for a high percentage of the total solids
weight, the ability to independently monitor such excipients is similarly
significant for issues such as quality control, manufacturing and
clinical performance. Unanticipated clinical effects can be a consequence
of previously unnoticed or changed distributions and types of excipients
that occur during manufacturing. For food systems, the development of
byproduct compounds due to thermal effects on saccharides provides
important insights into the adequacy and control of process manufacturing
steps. Such issues have been addressed for other products by H.-J. Kim,
U.S. Pat. No. 5,254,474 while others have focused on thermally generated
aldehydes in biotechnology applications, see C. M. Smales, D. S. Pepper,
and D. C. James, 2000, "Mechanisms of protein modification during model
antiviral heat-treatment bioprocessing of beta-lactoglobulin variant A in
the presence of sucrose," Biotechnol. Appl. Biochem., October, 32 (Pt. 2)
109-119. Detection of compounds linked to thermal effects on saccharides
in pharmaceutical manufacturing can also be significant where heat is a
factor in synthesis. Indeed, identification of nominal excipient levels
and distributions in complexes such as FHSC and SFGCS can verify that
proper synthesis conditions have been employed.

[0033]The present invention provides methods for separating hematinics
including SFGCS, FHSC and iron saccharates into at least two distinct
fractions. Furthermore, the methods are generally applicable to products
in which iron is supplied as an iron-saccharidic complex or an iron
delivery vehicle where iron is held in association with negatively
charged carboxyl groups derived from carbohydrates. Initially, one
fraction of the hematinic, identified herein as Fraction 1, comprises the
iron-saccharidic complex or iron-delivery active hematinic species, AHS.
Another fraction, identified herein as Fraction 2, comprises a mixture of
substantially all of the excipients that coexist with the AHS in the
original composition, e.g., as synthesized or as released. Excipients in
Fraction 2 can be, for example, hydrophilic organic or ionic compounds.

[0034]The solutes or suspended or dispersed components present in liquid
Fractions 1 or 2 can be used for detailed analytical study or
characterization purposes, and can be further concentrated or used as
starting materials in the development of new products. The present
invention discloses the separate, but related, analytical roles that
these fractions can play so as to facilitate the establishment of
benchmark reference standards that characterize sodium ferric gluconate
complex in sucrose, SFGCS, and ferric hydroxide sucrose complex, FHSC,
from their excipients. Furthermore, the present invention discloses
methods for preparing concentrated, lyophilized and optionally
reconstituted AHS. Separation of the various components is achieved
starting with the as-synthesized or commercially available hematinic with
its combined AHS and excipients in an aqueous (water) composition and
then selectively relocating or extracting the excipients so that they are
present in a substantially separate aqueous phase. After establishing
these two fractions, each can undergo detailed analytical
characterization, further purification if desired, and concentration to
satisfy analytical, synthesis or manufacturing goals.

[0035]Hematinics are a class of pharmaceuticals designed to convey
hematopoietically useful iron and Fraction 1 comprises from at least
about 75 to about less than about 100%; preferably; from at least about
80 wt. % to about 99.9 wt %; more preferably from at least about 90 wt. %
to about 99.9 wt. %; most preferably from at least about 95 wt. % to
about 99.9 wt. %; of the AHS intended for parenteral delivery, whereas
Fraction 2 comprises correspondingly low levels of the AHS, for example,
less than about 0.1 wt. % and substantially all of the excipients
originally present in the hematinic composition.

[0036]1. Substantially Separating the AHS from Coexisting Excipients.

[0037]At least one active hematinic species, AHS, can be isolated or
substantially separated from excipients present in an as-synthesized
hematinic or in commercially available pharmaceuticals characterized as
iron-saccharidic complexes. Such separation has been achieved by the
inventors herein as a consequence of their determining several
significant characteristics of hematinic compositions which comprise an
iron-saccharidic complex, including: (1) that it is necessary to increase
the Aw value of the AHS-containing phase or fraction; (2) AHS
display at least one detectable iron species with a formula weight of
from about 250,000 to about 3,000,000 Daltons or more; (3) manufacturing
and stability variations can result in more than one detectable
iron-containing species being present; (4) Fe(III)-containing species can
display different shapes, e.g., as measured by laser light scattering;
(5) Fe(III)-containing AHS have an electrical charge; and (6)
Fe(III)-containing AHS can have a detectable oxidation-reduction (redox)
potential indicative of the presence of ferric iron, (Fe(III), or ferrous
iron (Fe(II). Separation of AHS from its excipients into at least two
fractions may be achieved by using one or more of the following methods,
with or without preliminary pH stabilization in the range of about 6.0 to
about 8.0; preferably about 6.4 to about 7.8, more preferably about 6.6
to about 7.6 for example about 6.8 to about 7.4:

[0038]1. Electrokinetic migration where AHS concentration depends on
direct electrical current flow that causes electrically charged AHS to
deposit on a charged collection surface or within an aqueous volume in
which a charged surface is present separate from its excipients.

[0039]2. Electrokinetic-based membrane technology wherein a cathode and an
anode are placed in a water system separated by a semipermeable membrane
partition. A direct current applied over the membrane causes the
electrically charged AHS to concentrate on the appropriate electrode due
to attraction between opposing charges. Such AHS concentration on a
single electrode in one compartment allows the dialysis-mediated removal
of hydrophilic excipient carbohydrates through the semipermeable membrane
into the accompanying compartment. Preferred semipermeable membranes
include cellulose, cellulose acetate, cellulose ester or regenerated
cellulose. In order to maintain retention of the AHS in one compartment
the membrane has a preferred molecular weight exclusion size of about
90,000 to about 300,000; preferably about 150,000 to about 200,000.
Preferred conditions for the method include the use of distilled,
deionized water at a pH of about 7.5 to about 9.8; a pressure of about
1.0 atm; and a temperature of about 2° C. to about 50° C.
The rate of dialysis removal of hydrophilic excipients can be improved by
frequent changes in aqueous dialyzing fluid. The process is generally
described by Ficks Law, F=-DA(dc/dx), where F=the total flux; D is the
diffusivity of the species in the medium, e.g., water; A is the surface
area available for diffusion and dc/dx is the concentration gradient of
the excipients through the membrane.

[0040]3. Capillary electrophoresis technology that concentrates AHS from
coexisting excipients. Capillary electrophoresis, sometimes referred to
as capillary zone electrophoresis, relies on the introduction of an
electrically charged analyte within a fused silica capillary with size
dimensions of about 50 to about 75 microns in diameter by about 50 to
about 100 cm in length to which is applied a voltage of up to about 30
kilovolts. The differential electrokinetic migrations of charged
substances in the composition can be detected and recorded by a variety
of methods described herein, including UV-VIS, fluorescence and mass
spectroscopy. The final migration positions of the electrically charged
substances in the capillary can be documented as an electropherogram.
This method can be particularly useful owing to the substantial
electrical charge on the AHS within iron-saccharidic complexes.

[0041]4. Column chromatography, which is a particularly preferred process
for selective separation of AHS. Owing to the discrete formula weight,
size dimensions, shape and charge of the AHS in contrast to coexisting
excipients, AHS engage in different stationary phase interactions as a
liquid carrier or eluent (e.g., water) transports them over and/or
through a solid support. Thus, differential diffusion or migration rates
responsible for such separations reflect relative electrical charge
and/or size exclusion differences that retard or accelerate elution of
these substances through the chromatography system. Separations can also
be adjusted by modifying the porosity, electrical charge or adsorption
properties on the surface of the stationary phase. Such chromatographic
processes may be carried out at from about 3° C. to about
150° C.; preferably from about 15° C. to about 35°
C.; typically, 25° C. using aqueous or non-aqueous solvents
supplied to columns in a singular or multiple serial flow scheme.
Chromatographic partitioning of AHS may be carried out at any pressure
drop over the inflow and outflow of a column. The internal pressure of
the column may range from below atmospheric pressure to any pressure that
the column and stationary phase material can tolerate. Operational
pressures above about one atmosphere (0.1 MPa) are preferred, but
pressures up to about 10,000 pounds per square inch (69 MPa) may be
employed. Eluants supplied to the column can include any solvent or
diluent so long as AHS is maintained as an iron-saccharidic complex. Such
eluants include C1-C6 alkanols, ethanolamine,
dimethylsulfoxide, carbonyl-based solvents, dimethylforamide, water,
aqueous buffer solutions and various admixtures including
water-saccharide solutions. The use of from about 2.0 to about 25 weight
percent of a primary alcohol may be useful for controlling potential
microbial growth. Suitable stationary phase materials are commercially
available including porous silica, crosslinked polyglucans identified as
dextrans, crosslinked methacrylate polymers, copolymers of ethylene
glycol and methacrylate, crosslinked polystyrene, alumina, agarose gels,
cyclodextrins and cationic as well as anionic exchange packings may be
used. Particularly preferred stationary phases for column chromatographic
separation of AHS from its excipients in a hematinic composition is
crosslinked polyglucan or dextran available commercially in various
grades as Sephadex G-10, G-15 and G-25 (Amersham-Pharmacia Biotech.,
Piscataway, N.J.); and, a commercial column identified as GMPWXL
having a 13 micron particle diameter with pore sizes of from about 100 to
about 2000 Angstroms and a polymethylmethacrylate backbone (Tosoh Biosep,
Montgomeryville, Pa.). When employing solid stationary phase packing,
pore diameter in the range of from about 30 to about 9000 Angstroms is
preferred; more preferably from about 100 to about 8000 Angstroms. The
dextran stationary phases are particularly preferred for the bulk
separation of AHS, e.g., using low pressure chromatography; and
polymethylmethacrylate backbone is particularly preferred for analytical
characterization of the AHS, e.g., using HPLC.

[0042]The methods of the present invention can be used singularly or in
combination with each other in order to separate AHS that is primarily or
substantially responsible for the desired pharmacological action
attributed to FHSC or SFGCS. The substantially separated and,
consequently, purified AHS serves as the basis for establishing a primary
reference standard. In turn, the primary reference standard can then
provide a standard for use in monitoring manufacturing and pharmaceutical
quality control. If desired, the AHS can be subjected to further
purification using the methods taught herein. For example, where analysis
of a hematinic composition using HPLC in combination with, for example,
LLS and RI, shows the presence of a shoulder on, or a peak preceding the
AHS primary reference standard, a further separation method can be
employed. As described herein, such a shoulder or secondary peak may be
present in a hematinic composition as a consequence of departure from
preferred manufacturing conditions or as a result of storage of the
hematinic composition, particularly in the presence of hydrophilic
excipients. The further separation or purification process comprises
additionally separating the first to elute material from the preparative
chromatographic column using the HPLC analytical results or data obtained
directly from an LLS detector in combination with the preparative
chromatographic column. In this manner, a significant percentage or
substantially all of the undesirable aggregated AHS can be separated from
the preferred AHS. Where an initial non-AHS peak is present and
distinctly separate from the characteristic AHS primary reference
standard peak, substantially all of the aggregated material corresponding
to such a peak can be separated from the desired AHS, for example, in
Fraction 1. Where a shoulder appears on the primary reference standard
AHS peak, as will be further discussed below, separation of substantially
all of the undesirable, e.g., aggregated material, may necessarily carry
with it some of the preferred AHS or, conversely, not all of the
aggregated material may be removed. The extent of separation and
purification can be determined with reference to the data generated
according to the methods of the present invention. Therefore, in addition
to removal of substantially all of the lower molecular weight, primarily
hydrophilic excipients present in the hematinic compositions of the
present invention, the methods taught herein can result in an AHS that is
substantially purified as well with regard to aggregated iron complexes.

[0043]Applying the teachings of the present invention, a discrete active
hematinic species (AHS) can be separated from excipients supplied in
commercially available parenteral compositions characterized as
iron-saccharidic complexes; the separated material can serve as a
"primary reference standard." One method of separating the AHS from
coexisting excipients employs low pressure gel permeation chromatography
(GPC). In the present invention, low pressure refers to operation of the
chromatography column at about ambient pressure, including at a pressure
slightly above ambient as a consequence of pumping fluid to a packed
column to which are attached valved lines and, optionally, other
equipment, including one or more analytical instruments or detectors.
This technique can be used not only for analysis of the separated
materials, but also for producing bulk quantities of AHS for preparation
of new parenteral compositions. Preferably, the column packing comprises
epichlorohydrin-crosslinked polyglucans with demonstrated molecular
weight or size exclusion characteristics greater than about 5,000
Daltons; more preferably with size exclusion characteristics greater than
about 1,500 Daltons are preferred. Suitable equipment is available from
Amersham-Pharmacia Biotech, Piscataway, N.J. Furthermore, ion-exchange
gels with GPC properties and affinity chromatography columns are also
suitable.

[0044]The separated AHS of the present invention, substantially free of
excipients, generally has a high absolute molecular weight when measured
by the techniques described herein, e.g., HPLC in combination with LLS
and RI. Such high absolute molecular weights are typically greater than
about 25,000 Daltons and can be greater than about 30,000, 50,000, 75,000
or 100,000 to about 3,000,000 Daltons or more; for example, molecular
weights of about 200,000 to about 2,500,000; about 250,000 to about
1,000,000; or about 275,000 to about 850,000. These high molecular
weights are to be contrasted with the low molecular weights reported in
the literature for compounds intended for use in tablet form. Such low
molecular weight compounds are less than about 2200 Daltons, more
typically less than about 1700 Daltons and, in the present invention, are
separated from high molecular weight AHS when using, e.g., column packing
having exclusion characteristics of greater than about 5000 Daltons.
Consequently, as described herein, separation of high molecular weight
AHS from low molecular weight excipients distinguishes the AHS of the
present invention on the basis of molecular weights, even where such low
molecular weights are as high as 5000 Daltons.

[0045]The AHS is typically present in Fraction 1 and the excipients elute
thereafter in Fraction 2. Since material elutes from the column in a
continuous fashion, the reference to fractions is based on that portion
of material eluted from the column in which there is present
substantially all of the preferred or primary reference AHS that has been
substantially separated from its excipients. One method for marking such
a separation of eluted material is to observe where the LLS signal
closely approaches or returns to the baseline value for the mobile phase
following the appearance of the initial peak or peaks indicating the
presence of AHS and/or aggregated AHS (as further discussed below). In a
preferred method, a solvent reservoir supplies an aqueous-based diluent
or solvent by gravity or metered flow to a chromatography column of any
selected diameter or length provided that the length is at least twice
the diameter. The column can be constructed of glass, stainless steel,
polycarbonate or another material that is nonreactive with the
composition and diluents or solvents employed and able to contain a
stationary chromatographic support material, also referred to as a bed.
The bed typically comprises beads having suitable porosity but other
forms of the bed may be manufactured in situ within the confines of the
column, such as a poured-in-place porous polymer. In carrying out the
process of the present invention, initially an aqueous-based solvent (or
diluent), referred to as the mobile phase, is passed over as well as
through the interstices of a beaded, porous support bed. As the liquid
stream or eluate exits the column, it is conducted, e.g., through tubing,
to one or more detectors that analyze the stream in order to determine
its baseline properties. Detectors may be positioned to deal with a
series flow of eluate from one detector to the next or a split stream
flow that allows parallel, multiple detector monitoring. Such detectors
target eluate characteristics in terms of real time volumetric flow of
the stream. Suitable detectors are employed to measure and record such
stream properties as pH, electrical conductivity, electrochemical
reduction potential, refractive index (RI) and other useful analytical
properties. UV-VIS absorption (A) and refractive index (RI) are preferred
detectors for measuring the baseline properties of the mobile phase.

[0046]Injection of an iron-saccharidic complex, for example a commercially
available parenteral composition, into the aqueous stream leading into
the top of the packed column bed ensures that various constituents in the
composition will be distributed over and through the porous
chromatographic beads. Without wishing to be bound by theory, it is
believed that separation of the different sized chemical species
comprising the iron-saccharidic complex proceeds, for example, by a
sieving effect and, potentially, with hydrogen bonding interactions.
Chemical species larger than the pores are excluded from the pores and
they first exit the column in a relatively small eluate volume
(Ve1). As the flow of the sample continues through the column,
progressively smaller molecules can become entrained within the pores and
subsequently exit the column in a relatively larger eluate volume
(Ve2). Thus, large species such as AHS within the hematinic elute
first (Ve1) in Fraction 1 and smaller excipient chemical species
elute later (Ve2) in Fraction 2. Such a chromatographic separation
produces at least two components or fractions, the AHS and its
excipients, when applied to the initial parenteral volume of hematinics
including components such as SFGCS, FHSC. As will be described later,
larger sized byproduct or degradation species can also elute with or
prior to the AHS. The AHS separation is substantially complete so that it
is separated from original coexisting excipients, particularly
hydrophilic excipients, and including those excipients that may have
distinctive fluorescent properties. Consequently, the AHS is produced in
a high Aw aqueous fraction, Fraction 1, that is substantially free
of excipients and substantially all of the excipients are present in a
second fraction, Fraction 2, characterized by a low Aw condition.
Acid hydrogen-base binding (AH-B) dynamics within the column bed can
further increase the effective elution volume (Ve2) for excipients
from the column in addition to their size interactions with the
chromatographic bed. Acid hydrogen-base binding interactions are
generally discussed by Hodge, J. E. and E. M. Osman, 1976, Chapter 3, in
"Food Chemistry," O. R. Fennema Ed., Marcel Dekkar, New York, pp. 92-96;
and Zapsalis C. and R. A. Beck, 1985, "Food Chemistry and Nutritional
Biochemistry," Chapter 10, John Wiley & Sons, pp. 588-591 (each
incorporated by reference to the extent permitted). For purposes of the
present invention the term "substantially free" when used in reference to
the separated AHS being substantially free of excipients means that the
fraction containing the AHS includes from greater than 80 wt. %;
generally greater than about 85 wt. %; preferably greater than about 95
wt. %; more preferably greater than about 98 wt. %; still more preferably
greater than about 99 wt. % and most preferably greater than about 99.9
wt. % up to less than or equal to about 100 wt. % of the AHS eluted from
the column (in other words, there can be present trace amounts of
excipients and the amount of eluted AHS may not include a minor or trace
amount of AHS that may be retained in the column, tubing, detectors, or
lost during processing). Correspondingly, such excipients as are
originally present in the composition fed to the chromatographic column,
or possibly generated during processing, are contained in the subsequent,
excipient fraction, Fraction 2, and is equal in amount to the above
stated values subtracted from a value of 100 wt. %. For example, if the
AHS fraction comprises greater than about 99.9 wt. % AHS, excipients can
be present in an amount less than about 0.1 wt. %. Separation of AHS from
non-hematinic components, excipients, also typically separates the AHS
from sucrose that may have been added during the synthesis process or,
possibly, post-synthesis. To the extent that sucrose can be readily
separated from the AHS, it is to be understood that the AHS of the
present invention is distinct therefrom, although a chemical formula or
structural diagram may suggest that sucrose is present. Consequently,
with regard to the two commercially available hematinics, the separated
AHS in one instance corresponds to sodium ferric gluconate complex and,
in the other, to ferric hydroxide-sucrose complex, although the
commercial products may be identified as being "in sucrose." Since
sucrose is readily separated from AHS by the methods taught herein, for
the purposes of the present invention, sucrose is an excipient. However,
if desired, sucrose can be added to a composition that includes the AHS
of the present invention if its presence is considered to serve a useful
function, e.g., to modify the characteristics of a parenteral
composition.

[0047]The presence of the AHS, compared to the excipients, in the
chromatographic eluate stream can be observed using laser light
scattering, as described. Additionally, the elution profile of the AHS
and excipients can be detected by using one or more different types of
detectors whose output signals are simultaneously recorded for the eluate
stream. In a preferred method, one detector is employed that is sensitive
to wavelength (λ) using a UV-VIS absorbance detector and another is
a concentration sensitive refractive index (RI) detector. The dual,
independent output signals from these detectors are processed and
separately recorded as two independent ordinate axes (y-axes) against a
common abscissa (x-axis) expressed in units of cumulative eluate volume
(e.g., milliliters) or slices (i) as discussed earlier. This method can
identify where so-called Fraction 1, comprising substantially all of the
AHS effectively ends and Fraction 2, comprising substantially all of the
excipients, effectively begins. Experimental evidence demonstrates that
the fractions are sufficiently separated in time so that the AHS can be
obtained substantially free of excipients.

[0048]According to the Beer-Lambert Law, light absorbance is related to
the concentration of a specific light-absorbing species. The absorbance
(A) of a light absorbing species is expressed as:

A=εbc Equation 5

[0049]where c is the concentration of a light absorbing species in moles
per liter (M/L); b is the light path in centimeters (cm) through the
light absorbing species; and, ε is a proportionality constant
known as the molar extinction coefficient. This proportionality constant
is unique to a light-absorbing species at a given wavelength of light.
This fundamental law accounts for the fact that light absorbance for a
light absorbing species at a specific wavelength (λ) converts into
a quantitative measure of its molar concentration. Hence, positive
increases in absorbance at a prescribed wavelength for an analyte
corresponds to increases in its molar concentration. For monitoring the
AHS of SFGCS or FHSC in a chromatographic eluant stream, 435 nm is
preferred because of high extinction coefficient for that wavelength,
i.e. 6.590 log10, for iron-saccharidic complexes.

[0050]For the preparation of substantially excipient-free AHS using
chromatographic separation, the A430 nm value is tabulated against
column eluate volume. This can be transformed into a chromatographic
elution profile where A430 nm ordinate (y-axis) values and eluate
volumes (x-axis) are plotted against each other. As discussed, the larger
AHS elutes from the chromatographic column before smaller excipients. So
long as a change in absorbance (ΔA) with respect to changes in
eluate volume (ΔV) or ΔA/ΔV consistently shows a
positive ratio (+ΔA/ΔV) above a solute-free baseline eluent
signal, AHS concentration increases in eluate volume concentration. For a
negative ratio (-ΔA/ΔV) the opposite is true. Where the
ΔA/ΔV ratio makes a transition from (+) to (-) the
chromatographic elution of the AHS is maximized as a "chromatographic
peak." From this point onward, as the chromatographic profile continues
to display a negative ratio (-ΔA/ΔV) the AHS concentration
diminishes in the eluate volume. This is true for AHS concentrations so
long as the A430 nm continues to asymptotically decrease and
approach an A430 nm value of about 0.0. As this measurement signals
an end to the elution of the AHS, the concentration sensitive RI detector
begins to respond to increasing amounts of hydrophilic and other
excipients included in the eluate. As above, a positive (+) change in the
ratio of RI with respect to changes in volume (+ΔRI/ΔV),
signals increasing concentration of excipients. At the eluate collection
volume where ΔA/ΔV still displays a negative ratio
(-ΔA/ΔV) asymptotically approaching a value of about 0.0 A
and RI-detector begins to signal a +ΔRI/ΔV slope, this marks
the effective boundary where Fraction 1 substantially ends and Fraction 2
effectively begins. Fraction 1 comprises the AHS in a high Aw
environment and Fraction 2 comprises the hydrophilic and other excipients
in low Aw environment. In this manner, an elution profile is
determined.

[0051]The eluate boundaries that define the separation where Fraction 1
substantially ends and Fraction 2 effectively begins can alternatively be
determined by measuring the percent spectral transmission of the AHS in
column eluate at 430 nm. A suitable instrument for this purpose is a
"ColorQuest XE" system manufactured by Hunter Associates Laboratory,
Inc., Reston, Va. Increases in the light transmission of the eluate as
the AHS present in the eluate asymptotically approaches zero percent, can
indicate substantial separation or an end to elution of the AHS. A
subsequent increase in RI-response of the eluate stream marks the
beginning of the eluate volume comprising excipients. In a further
alternative embodiment, a demarcation between where Fraction 1
substantially ends and Fraction 2 effectively begins can be determined by
measuring the A620 nm anthrone-based absorbance of the eluate
stream. Since both the AHS and the excipients have notable dextrose
equivalent (DE) absorbency, the detectable A620 nm DE-value will be
minimal between Fraction 1 and Fraction 2. The principles that underscore
this analytical concept have been described by R. Dreywood, "Qualitative
Test for Carbohydrate Material," Indus. and Eng. Chem., Anal. Ed., 18:499
(1946); J. E. Hodge and B. T. Hofreiter, "Determination of Reducing
Sugars and Carbohydrates," Methods Carbohydrate Chem., 1:384-394 (1962);
and more recently by C. Zapsalis and R. A. Beck, "Food Chemistry and
Nutritional Biochemistry," Chapter 6, John Wiley & Sons, pp. 353-354
(1985) (each of the disclosures of which are incorporated herein to the
extent permitted).

[0052]The methods of the present invention allow for preparation of large
production batches (for example, from greater than about 500 milligrams
to greater than or equal to about 1.0 gram; or from about 1.0 gram to
about 10 grams or more; from about 1.0 gram to about 100 grams or more;
from about 1.0 gram to about 1 kg or more; for example, if desired,
hundreds or even thousands of kilograms can be produced by the present
method) or small analytical samples (from about 5.0 to about 500.0
milligrams) of the AHS present in iron-saccharidic complexes of any type
can be prepared. Since the AHS is the preferred component of complexed
iron present of these parenteral compositions, the ability to separate
the AHS forms the basis for the primary reference standard. Separation of
Fraction 1 permits further detailed chemical and/or structural analyses;
such methods are described elsewhere herein. However isolated, the AHS,
e.g. Fraction 1, as well as the excipients, e.g., Fraction 2, may be
further concentrated for detailed study.

[0053]The present invention is also suitable for production of small scale
amounts of AHS (from less than about 1.0 mg to less than about 5.0 mg),
preferably by way of high pressure (or high performance) liquid
chromatography (HPLC). An appropriate chromatographic solid support can
be used to separate the AHS from its excipients. This results in AHS
transfer into a hydrodynamic volume within the column that displays a
high Aw, e.g., approaching a value of 1.0. The excipients are
transferred into a low Aw hydrodynamic volume having a low Aw,
e.g., less than the value of the AHS-containing phase. The operational
principle for this method is similar to that of the low pressure
chromatographic method described above, but the stationary column bed
materials for the HPLC method are more finely divided so as to withstand
pressures of from about 5,000 to about 10,000 pounds per square inch
(about 35 to about 69 M Pa). This results in slower flow rates of eluate
from the column, but it is more than compensated for by the high
hydrostatic pressure. A silica-based stationary bed that depends on
adsorptive-desorptive analyte separation phenomena can be used to produce
a separation of the AHS from its excipients. Such silica-based separation
performances however are difficult to control and require accurate
preparation of an azide-containing aqueous mobile phase; consequently,
they are subject to greater analytical errors. Furthermore, shedding of
particulates from silica-based beds complicates or can preclude effective
use of a LLS detector. Thus, a polymeric HPLC column, such as a
GMPWXL brand manufactured by Tosoh Biosep, Montgomeryville, Pa.,
that employs an aqueous mobile phase is preferred over silica-based
columns. Such HPLC analysis of, for example, a commercially released form
of iron-saccharidic complex requires preliminary 0.02 micron filtration
through, for example, an Anotop 25 brand inorganic membrane (Whatman,
Maidstone, UK).

[0055]Analysis for iron reveals that more than about 90% of the iron
intended for hematinic purposes resides in the high Aw fraction
designated as Fraction 1 above, generally from at least about 75 wt. % to
less than about 150 weight %; preferably from about 80 wt. % to about
99.9 wt. %; for example, from about 90 wt. % to about 99.9 wt. %; from
about 95 wt. % to about 99 wt. %; and excipients reside in the low
Aw fraction designated as Fraction 2. Iron atoms in Fraction 1 can
be quantified by atomic absorption spectroscopy (AAS) but iron
quantification by AAS alone is not determinative of hematinic
functionality of the products of the present invention.

[0056]The relevant characteristic of a non-dextran hematinic of the
present invention is based on its ability to deliver a physiologically
tolerable or benign source of ferric iron, Fe(III), preferably via
parenteral means. This ferric iron composition is a parenterally
acceptable species that resembles properties of an association colloid.
An association colloid is a reversible chemical combination due to weak
chemical bonding forces wherein up to hundreds of molecules or ions
aggregate to form colloidal structures with sizes of from about 1 to
about 2000 nanometers or larger. Such colloids of ferric ions interacting
with saccharidic compounds exhibit directional migration in an electric
field in addition to optical activity identified by laser light
scattering (LLS). LLS properties relevant herein relate to the Tyndall
effect where an incident light beam (Io) passing through a colloid
emerges from it at a 90° angle to its original path. Light
scattering only occurs if the light interacts with macromolecules such as
starches, proteins or other colloidal species where the wavelength of
incident light approaches size dimensions of the molecules. Light
scattering can occur as destructive interference where the scattered
wavelengths interact to cancel each other out or by constructive
interference where two wavelengths of light reinforce each other.
Mathematical evaluation of LLS data permits size and shape evaluations of
various colloidal species. Size, for example, may be estimated in terms
of molecular weight for a single molecule or the formula weight for a
multi-molecular or ionic aggregate. The weight expressions in either case
represent the sum of atomic weights of all atoms present in such
structures. The structural diversity of most aggregates or molecules such
as polymers is such that they exist as a frequency distribution of
varying weights, typically expressed as an average or mean molecular
weight distribution (MWD). Apart from size, colloidal shape can have
important implications. For example, if its shape is that of a thin rod,
a random coiled structure or a sphere its interaction with other
molecules or structures can vary. LLS, including multi-angle laser light
scattering (MALLS) or low angle laser light scattering (LALLS), combined
with one or more methods of HPLC-integrated detector analysis can be used
for evaluating iron-saccharidic complexes. For purposes of the present
invention, reference to LLS should be understood to include MALLS, the
latter being a preferred type of detector. The use of LLS measurements
herein provides a superior and preferred analytical method for
characterizing an iron-saccharidic complex that is normal, in other
words, represents the preferred AHS resulting from suitably controlled
synthesis, or one that displays evidence of decay or degradation
products. The fundamental mathematical relationships and operation of
HPLC in combination with laser light scattering and refractive index
detectors for the characterization of macromolecular structures and
association colloids has been reported. (see P. Wyatt, Light scattering
and absolute characterization of macromolecules, Analytica Chimica Acta.
(1993) 272:1-40; incorporated by reference to the extent permitted). As
discussed herein, such techniques are applicable to iron-saccharidic
complexes comprising AHS.

[0057]Acquisition of LLS data is particularly useful for identifying AHS
in saccharidic-iron complexes, particularly where a benchmark analytical
reference standard is required. LLS data can be obtained from an
individual batch based on an isolated sample or LLS data can be obtained
using in-line analysis of an eluate stream from a continuous process
containing the AHS following its liquid chromatographic isolation. In
other words, once a saccharidically bound ferric iron complex has been
produced and examined by LLS alone or in combination with other methods
such as those disclosed herein, batch-to-batch comparisons of
manufacturing continuity can be routinely verified in terms of formula
weight as well as morphology of the pharmacological species.
Characterization of AHS size and shape features can be used to monitor
AHS manufacturing processes, AHS product quality and AHS stability by
using LLS algorithms that are integral with the operational software of
LLS enhanced HPLC. Specifically, as taught herein, size and dimensional
attributes for so-called normal AHS (e.g., primary reference standard
material or the preferred AHS) and so-called abnormal AHS (e.g., degraded
or aggregated AHS) structures are determined from a standard Debye plot
generated by operating software such as ASTRA (Wyatt Technology Corp.,
Santa Barbara, Calif.). The details of such an application described in
P. Wyatt, Light scattering and absolute characterization of
macromolecules. Analytica Chimica Acta, 272:1-40 (1993), incorporated
herein by reference to the extent permitted. Preferred AHS has been
observed to have a generally spherical shape and to be about 10
nanometers or smaller in size, whereas degraded or aggregated AHS, such
as material that elutes from a chromatographic column before the
preferred AHS, tends to be greater than about 10 nanometers in size, for
example from about 10 to about 30 nanometers or more, for example, from
about 20 to about 30 nanometers or more. Size and shape dimensions can be
relevant factors for physiological and metabolic tolerances as well as
for their tissue disposition of a hematinic. Thus, the application of LLS
methods is preferred for routine characterization (including after
manufacturing and during storage) and manufacturing monitoring for the
hematinics of the present invention.

[0058]Laser light scattering of AHS provides characterization parameters
in terms of absolute formula weight or absolute molecular weight.
However, since the AHS is not a monomolecular species, but instead
behaves as an association colloid, the use of absolute molecular weight
is particularly preferred. Such LLS-based molecular weight measurements
are preferred over other methods of weight estimation that provide a
so-called relative molecular weight. Relative molecular weight
measurements of the AHS do not rely on the size and shape dependent
physical interactions of light with an AHS as a basis for estimating its
weight. Instead, relative molecular weight measurements rely on standard
methods of size exclusion chromatography (SEC), also sometimes referred
to as gel permeation chromatography (GPC). Both SEC and GPC are
hereinafter used interchangeably. In GPC analysis, the effective size of
a macromolecule or aggregate such as an AHS, not its formula weight,
determines its elution volume and exit from a calibrated chromatography
column. Hence, the relative elution and transit of the AHS through a GPC
column relative to a series of calibration standards in a narrow band
above and below the expected weight of the AHS provides the basis for
assigning a relative formula weight to the iron-containing complex. Using
this concept, a concentration sensitive detector such as a refractive
index (RI) detector would be used for detection of the AHS and its
calibration standards as they elute from a column. As a concentration
sensitive detector, RI-measurements rely on the changes in the indices of
light refraction (n) as analyte (for example, solute) concentrations (c)
increase or decrease in their respective flow through the RI-detector
cell. At evenly spaced time intervals, ratios of such changes in
refraction and concentration are recorded as dn/dc values. Each dn/dc
value is unique to a specific analyte or solute and the dn/dc value
characteristic of one substance is not an a priori value universally
applicable to any other specific substance. Such dn/dc values recorded
for the elution of known calibration standards and unknown analytes can
be acquired and recorded at precise time intervals. Each dn/dc
measurement in the overall elution profile of a liquid chromatography
column is referred to as a slice (i). The slice number, its elution time
or elution volume can be used to reference this slice. Elution time of a
slice multiplied by the flow rate of the eluent to the column gives the
elution volume. Records of slice numbers in terms of volume or number do
not compromise the general significance of this data-recording concept.
Thus, a plot of dn/dc (ordinate) versus slice numbers (abscissa) over the
course of a GPC measurement gives an elution profile for calibrating
standards with respect to any unknown substances that may be totally
unrelated to each other, yet they have do/dc-detectable masses suitable
for instrumental monitoring.

[0059]Because standard GPC procedures do not recognize significant
physical interactions common to all large molecular or colloidal
structures, GPC weight evaluations for the AHS present in SFGCS, FHSC or
other similar hematinics, can be susceptible to analytical errors.
Furthermore, RI-based GPC methods are unable to characterize the shape
and topological features of the AHS including, for example, structural
branching. Also, estimations of relative formula weight using
RI-dependent GPC methods are also subject to data variations, are
sensitive to minor analyst errors and completely insensitive to
dimensional variations of species within association colloids that have a
high density. For association colloids, and FHSC and SFGCS in particular,
these features are important because nonsaccharidically aggregated iron
atoms can coexist along with the desired iron-saccharidic complex
containing the AHS. Thus, it is important to recognize that RI-based GPC
can provide relative formula weight values for analytes of AHS with
reasonable accuracy and reasonable precision, but the method overlooks
other important characteristics of AHS structure.

[0060]Based on well-known principles of light scattering physics,
isotropic particles with small dimensions such as macromolecules and
colloids, interact with light so as to permit the calculation of their
absolute weight and shape. This is true for the AHS contained in FHSC and
SFGCS. Unlike GPC-RI based analysis, the absolute formula weight for a
colloidal species can be determined independent of any calibration curve
that depends on pre-established graded molecular weight standards (M. J.
Vold and R. D. Vold, 1964, "Colloid Chemistry," Reinhold, NY; and
Zapsalis, C. and R. A. Beck, 1985, "Food Chemistry and Nutritional
Biochemistry," Chapter 8, John Wiley & Sons, pp. 507-547 hereinafter,
Zapsalis and Beck, 1985) (each incorporated by reference to the extent
permitted). It is established that the intensity of light scattered
(I.sub.θ) by a particle through an angle of θ depends on the
intensity of the incident light beam (Io), the light path distance
(γs) through the light scattering volume and the
polarizability (α) of the particle. For unpolarized light the
equation is

R.sub.θ(1+cos 2θ)=I74
γs2/Io=8π4/(1+cos 2θ) Equation (1)

[0061]The term R74 (1+cos 2θ) is the basis for the Rayleigh
ratio. Extension of this light scattering measurement to very dilute
solutions of particles with sizes smaller than the wavelength of incident
light (Io) gives the expression

R.sub.θ=2π2ηo2[(η
ηo)/C]2/Lλ4CM=K*CM Equation (2)

where R.sub.θ becomes the Rayleigh ratio (R); η is the
refractive index of a solution containing particulate species,
ηo is the refractive index of a solvent without particulate
species; C is the concentration of solute in terms of mass per unit
volume; M is the molecular or formula weight respectively for a molecule
or particulate species; L is Avagadro's number; λ is the wavelength
of light; and
K*=2π2ηo2[(η-ηo)/C]2/Lλ.sup-
.4 is the optical constant. This expression provides a basis for
establishing the Rayleigh ratio (R) or that fraction of incident light
(Io) scattered by a particle when a wavelength (λ) of incident
light is comparable to or larger than the size of some particulate
species. Accordingly, R is related to formula weight of an analyte such
as the AHS in SFGCS, FHSC and other iron-saccharidic complexes by the
physics of light scattering phenomena and not relative comparisons to GPC
calibration curves based on unrelated materials. Independent of
instrument detector geometry for detecting the Rayleigh ratio (R) when
incident light (Io) interacts with particulates, a relationship
stands where

R=K*CM Equation (3)

[0062]Consistent with in-stream eluate measurement concepts detailed
above, where do/dc values are acquired for GPC-RI based systems, Rayleigh
ratio (R) measurements are acquired for each slice in other words "i",
Ri, and the Raleigh ratio is simply the product of each slice's
concentration (Ci), molecular weight (Mi) and the optical
constant (K*) or,

Ri=K*CiMi Equation (4)

[0063]Light scattering also allows for characterization of structural
consistency of the AHS in iron-saccharidic complexes based on dissymmetry
ratios between light scattered at some forward angle θ and that
light scattered at its supplementary angle
180-θ(I.sub.θ/I180-θ). Of all possible light
scattering angles, about 45° and 135° serve as instructive
reference points. When a plot of I.sub.θ/I180-θ
(ordinate) versus L/λ (abscissa) is constructed, the structural
dissymmetries for spheres, rods and random coils are readily determined
by way of reference to Zapsalis and Beck, 1985, p. 535.

[0064]If the AHS is substantially isolated, such as Fraction 1 discussed
above, it may be harvested in (a) bulk production amounts or (b) small
analytical amounts typical of that found in liquid chromatographic eluate
streams. In either case, LLS methods offer a preferred analytical method
for establishing a routine reference standard for any iron-saccharidic
complex in this pharmaceutical class.

[0066]Furthermore, the ability to separate the AHS into at least two
fractions, Fraction 1 (high Aw fraction), and Fraction 2 (low
Aw fraction), also permits independent analysis of excipients that
are concentrated in Fraction 2. Discriminative excipient fingerprints can
be used to characterize the AHS as well as contribute to product
identification and monitoring quality control and assurance. The
occurrence and verification of typical or expected excipients supports
standardization and monitoring of manufacturing. This improves the
likelihood for favorable patient use in clinical settings where the
hematinic is administered. Since excipients can account for over 75
percent of the total solids in typical commercial parenteral
compositions, excipient verification and analysis can be an important
matter. Moreover, since thermally treated saccharides are known to
produce identifiable chemical markers that reflect their processing
history, these as well as surplus reactants and byproducts developed
during hematinic manufacturing (for purposes of the present invention,
all such materials are generally included in the term "excipients") can
be used to monitor and characterize both the hematinics and processes
used to produce them.

[0067]There are at least three possible manifestations wherein ferric ions
interact or exist in hematinic iron-saccharidic complexes. Firstly, the
preferred principal agent, namely the AHS, behaves as an association
colloid and it is the desired iron-saccharidic delivery vehicle.
Secondly, high formula weight aggregates can develop from the AHS and
they may also be detected. Thirdly, iron can exist as a complex with
surplus saccharidic reactants and/or byproducts of synthesis reaction
steps. This form of iron can be found with hydrophilic excipients in
Fraction 2. It is particularly preferred that parenteral compositions be
monitored, as well as Fraction (2), for (a) iron content, (b) residual
amounts of saccharidic reagents, and (c) evidence of thermally-dependent
synthesis reaction byproducts. A particularly preferred method for
monitoring such analytes includes the use of liquid chromatographic
analysis with refractive index (RI) and in-line eluate stream detection
using at least one of the following: laser light scattering (LLS),
electrochemical detection (ECD), photodiode array (PDA) based UV-VIS
spectrophotometry, infrared (IR) spectroscopy, and liquid chromatography
coupled with mass spectrometry (LC-MS), and, optionally, one or more of
the analytical and characterization methods described above. Whereas RI
is a concentration sensitive detector that allows quantification of
excipient saccharides, ECD detectors respond to metal and
nonmetal-containing compounds having characteristic electrochemical
oxidation-reduction potentials and UV-VIS based PDA analysis permits
detection of thermally produced saccharidic derivatives in addition to
low formula weight iron-complexed species.

[0068]The methods of the present invention further include methods for
characterizing the AHS and its coexisting excipients. Application of HPLC
to a previously prepared sample of Fraction 1 comprising AHS using the
polymeric column described above, in combination with in-stream dual RI
and LLS detectors of the HPLC eluate provides the results illustrated in
FIG. 1. This figure shows the separated and purified chromatographic
signature for the AHS present in Fraction 1 and, furthermore, that it is
free of excipients. Such an HPLC elution signature is a preferred result
for a manufactured hematinic comprising an AHS, for example a product
released in a sealed glass ampoule. FIG. 2 illustrates the
chromatographic signature for four excipients present in Fraction 2
obtained from the same sample that provided Fraction 1. In this test, a
small amount of the AHS from Fraction 1 was intentionally introduced into
Fraction 2 so that relative positions of the excipients to the AHS could
be observed. It is worth noting in FIGS. 2 and 3 that excipients are
better monitored by an RI detector because RI is a concentration
sensitive property whereas an LLS detector is mass sensitive and responds
better to AHS than to excipient carbohydrates. These effects are clearly
shown in FIG. 2 where only a trace amount of pure AHS was added as an
internal benchmark for relative measurement of excipient elution
progress. RI in this case fails to sense any occurrence of purified AHS
yet the LLS signal records its presence as an analytical species. FIG. 3
further illustrates that the LLS and RI-based HPLC method using a
polymeric column can be used to analytically separate the AHS from its
excipients. That is, the AHS component is separated into a high Aw
hydrodynamic volume, while at the same time the coexisting excipients
that originally accompanied the AHS are substantially separated into
hydrodynamic column volumes that elute after elution of the AHS. These
results are consistent with those in FIG. 1 for the primary reference
standard previously isolated as a discrete entity whereas FIG. 3
represents results for a multicomponent initial composition (AHS and
excipients). This confirms that the methods of the present invention can
be used to monitor hematinic compositions such as those produced
commercially that comprise AHS and excipients.

[0069]Conditions such as instability and aging of the AHS and
manufacturing variability can be monitored in iron-saccharidic complexes
using HPLC analysis equipped with at least LLS as one detector mode.
Furthermore, LLS-based HPLC used in conjunction with a concentration
sensitive detector, such as RI, can be used to monitor structural
variations in the nominal chromatographic peak signature for an AHS. Such
variations can be seen in the chromatograph in FIG. 4, particularly when
compared to that produced by the primary reference standard in FIG. 1 or
the separated mixture in FIG. 3. Similarly, FIG. 5 shows the presence of
an altered AHS peak in another manufacturer's hematinic product. It is
noted that different detectors can provide different information, for
example, the LLS-chromatographic detector and the concentration sensitive
RI-detector. The concentration sensitive RI-detector senses and records
analyte concentration but not its mass, which is independently documented
by the LLS-detector. Thus, the LLS-based detector can sense increasing
mass of a species formed as a consequence of, e.g., complexing or
cross-linking of AHS, that elutes before the AHS primary reference
standard. Since the AHS, as illustrated in FIG. 1, is the desired
hematinic substance, the species shown in FIGS. 4 and 5, represent even
higher formula weight byproducts that are believed to arise from the AHS.
The new peak appears to develop at the quantitative expense of the
preferred AHS.

[0070]The methods of the present invention can be used to monitor storage
stability of hematinics comprising an AHS. Like most organic molecules,
AHS is subject to structural instability, such that it can degrade or
transform, even within sealed glass delivery ampoules used for
hematinics, to give new species. Such structural transformations of an
AHS are time dependent and may also be promoted by temperature. Evidence
of structural AHS transformation over time is evident for a series of LLS
and RI-based HPLC profiles for iron-saccharidic complexes stored in
sealed glass delivery ampoules at room temperature in the dark for 6, 12,
22 and 25 months after manufacture. The pertinent chromatographic
profiles for these products correspond, respectively, to FIGS. 6, 7, 8
and 9. Evidence of structural change is apparent when comparing the
unsymmetrical AHS peaks in these figures against the preferred and
symmetrical primary reference standard peak exhibited by the AHS in FIG.
1. The new entity indicative of AHS degradation, and detected by LLS,
elutes from the column before the primary reference standard or AHS,
which also can be seen in the figures. The new entity has features of a
very dense, high formula weight structure, conceptually similar to "BB
shot". These characteristics are determined based on the Debye plot
generated by processing the light scattering data using the ASTRA-brand
software incorporated in the LLS equipment, referred to earlier (Wyatt
Technology Corp., Santa Barbara Calif.), and the methods and calculations
described in the 1993 Analytica Chimica Acta journal reference, also
referred to above. Debye plots permit the acquisition of specific LLS
data which, in conjunction with root mean square (rms) or radius of
gyration (Rg) measurements, permit size determinations of molecular
species or colloids. For FIGS. 6-9, the rms-value indicating particle
diameter sizes gives an average value equal to or greater than 20 nm. The
corresponding rms-value for the chromatographic signature of AHS in FIG.
1 is less than or equal to 10 nm; well below the practical detection
limit of LLS. Thus, the methods of the present invention can be used for
monitoring the quality and storage stability of a hematinic composition
comprising AHS.

[0071]Commercially produced hematinics can be monitored for the AHS, as
well as for excipients, during the manufacturing process, at the
conclusion of manufacturing, e.g., at the time of packaging, and after
manufacture, e.g., if the product is stored. Similarly, an
iron-saccharidic complex composition comprising the AHS after separation
of the excipients, such composition in the form of an aqueous composition
or after drying, e.g., by freeze drying, can also be monitored in the
same manner. The iron-saccharidic complex comprising the AHS can be
monitored not only during manufacturing, but also as it is stored from
shortly after manufacture, such as from about one week thereafter, as
well as after a moderately short storage period of about 6 months to for
as long as about five years or more after manufacture; extended storage
can be from about 1 year to about five years; or from about 1 year to
about 3 years. In each instance, the AHS can be monitored by comparison
of its properties, including various analytical properties as discussed
above, e.g., the chromatographic signature obtained using HPLC in
combination with LLS and RI to a primary reference standard.

[0072]As described above, the smaller LLS-detectable peak resulting from
degradation or modification of the AHS originally present is an extremely
high formula weight substance, identified in FIGS. 6-9 as an AHS
aggregate peak (AHSAP). The substance responsible for this characteristic
AHSAP has a formula weight in the range of from about 350,000 to about
3,000,000 Daltons or more based on light scattering, but
ultracentrifugation and other methods can also be used to establish the
high formula weight of this species. It is believed that the AHSAP is
unrelated to excipients normally occurring as a consequence of the AHS
synthesis or manufacturing process. The AHSAP appears, instead, to be
related to an aging phenomenon, but it may also result from significant
departures from preferred manufacturing conditions. The methods of the
present invention can be used to identify the levels of quantitative
tolerance of the AHSAP in a parenteral hematinic product. The occurrence
and detection of an AHSAP at any time after manufacture (AHSAPTAM)
of an iron-saccharidic complex can be compared to the total quantitative
HPLC signal for the AHSAP detected after a prescribed storage period of
the hematinic while exposed to defined conditions (AHSAPTOTAL). By
way of example, the storage period could cover any convenient period of
time, for example from about 6 months to about 10 years or longer;
alternatively from about 1 to about 8 years; or from about 1 to about 5.0
years. If, for example a 5 year period is used, at 5 years the
AHSAP5yrTOTAL would provide the basis for establishing the maximum
acceptable decomposition or modification of the measured characteristics
of the hematinic after its release. In other words, by the time 5 years
had elapsed the AHS may have decayed into pharmacologically useless iron
aggregate and compositional residues distinctly unlike the intended
embodiment of the initially released iron-saccharidic complex for
parenteral use. Thus, the AHSAPTAM quantified at any time up to
AHSAP5yrTOTAL, expressed as an occurrence ratio,
[AHSAPTAM]/[AHSAP5yrTOTAL], can provide a time-dependent
stability ratio or index for gauging hematinic quality and actual percent
composition of intact AHS remaining in a hematinic product. There is
currently no such standardized way to quantitatively address aging and
decay of iron-saccharidic complexes once they are released into commerce.
It should be appreciated that the occurrence ratio is not limited to use
of the 5 year elapsed time interval, but, rather, as noted above, it
applies to any convenient elapsed time interval selected. It can be
useful to gauge iron-saccharidic complex aging and stability against
defined conditions where, for example, 50 percent of the iron-saccharidic
complex remains in its original form at the time of manufacture or
release into the marketplace, as compared to iron other than
iron-saccharidic complex; for example free or unbound iron or iron
aggregate, thereby allowing determination of an iron-saccharidic complex
half-life. As the [AHSAPTAM]/AHSAPTOTAL] ratio approaches a
value of about 0.5, this 0.5 ratio-value can serve as a pharmacokinetic
index and guardrail to ensure that the manufactured product will have at
least 50% of its iron-saccharidic complex still intact, as intended for
initial release. Although the basis for quantitatively gauging the shelf
life of hematinics here cites a value of 50% survival for
iron-complexation in an AHS, practical quality standards higher than 50%
are more desirable; as a practical matter, from about 0.5 to about 0.98;
preferably from about 0.75 to about 0.95; more preferably from about 0.80
to about 0.99; for example, any single value between about 0.5 and less
than or equal to about 1.0 (and correspondingly, for iron other than
iron-saccharidic complex, AHS, values of from 0.02 to less than about
0.5; from about 0.05 to about 0.25; and from about 0.01 to about 0.20;
for example, any single value from equal to or greater than about 0 to
less than or equal to about 0.5) may be established by a standards
setting organization or by the manufacturer. Such an established ratio of
[AHSAPTAM]/AHSAPTOTAL] is particularly useful for purposes of
indexing, warranting or standardizing the clinical efficacy, performance
and safety of such hematinics. Prior to the present invention, there was
no basis for assigning quality compliance standards to iron-saccharidic
complexes using standards that are generally applicable to drugs. As
described herein, the use of the HPLC chromatographic method, preferably
including LLS and RI-based detectors allows implementation of such
indices. Furthermore, the ability to isolate the AHS present in these
iron-complexes reinforces the practical application of the method.

[0073]Since the AHS, referred to as the primary reference standard for
iron-saccharidic complexes, can begin to degrade shortly after synthesis
or manufacture it can also be useful to establish a secondary reference
standard. In practical terms, the secondary reference standard is based
on the relative occurrence of iron aggregate derived from the active
hematinic species compared to the total amount of iron aggregate capable
of being released by the active hematinic species under set conditions
over time. The measurement of iron aggregate is justified for
establishing such benchmarks of active hematinic species integrity and
stability because detectable iron aggregate levels are formed at the
expense of the primary reference standard. The
[AHSAPTAM]/[AHSAPTOTAL] ratio plotted versus time after
iron-saccharidic complex manufacturing or commercial release, provides an
index of product storage stability. The time required to reach an
arbitrary or performance-related ratio, e.g., 0.5, based on the HPLC
signal quotient of [AHSAPTAM]/[AHSAPTOTAL] can be especially
useful, although any ratio can be selected as a guardrail to ensure
active hematinic species product quality. Whatever ratio is selected to
serve as a minimum acceptable standard to monitor clinical efficacy,
utility and safety based on historical use will set the compositional
standard for the secondary reference standard. Such a primary reference
standard or a secondary reference standard, can be prepared as practical
analytical standards for use in monitoring inter- and intra-laboratory or
manufacturing performance and as a product quality index and product
standardization tool.

[0074]The separated AHS-containing composition, Fraction 1, comprising the
primary reference standard can be dried for extended storage and
reconstituted for parenteral use and additional study.

[0075]Dried and/or reconstituted AHS can be stored for purposes of
advanced analytical characterization, for example, in order to establish
more definitive chemical criteria, as well as archiving AHS samples for
future reference. Storage of the separated and lyophilized AHS is
important because iron-saccharidic complexes are subject to
destabilization and decomposition following their synthesis, particularly
when such complexes remain in a diluent or liquid, particularly aqueous,
carrier. In contrast, the dried AHS can be stored for extended periods of
time, preferably in a moisture-free environment, including sealed
containers. Furthermore, the dried, stable complex can be conveniently
transported and reconstituted when needed at the point of use, thereby
further extending its stability until just prior to use. For example, the
dried AHS can be sealed in moisture proof containers such as metal foil
pouches or glass containers, and stored at ambient temperature (about
20° C. to about 25° C.) or below for extended periods of
time. For example, the dried complex can be stored from shortly after
manufacture, such as from about one week thereafter, as well as after a
moderately short storage period of about 6 months to for as long as about
five years or more after manufacture; extended storage can be from about
1 year to about five years; or from about 1 year to about 3 years. During
such post-manufacture storage, the AHS can be monitored for stability by
comparison of the analytical properties, e.g., the chromatographic
signature obtained using HPLC in combination with LLS and RI, of a
reconstituted sample to a primary reference standard.

[0076]The isolated AHS (Fraction 1), as initially made or at any
particular time thereafter, can be freeze dried (lyophilized) and
reconstituted for ease of storage and transportation as well as for
additional study. As a prerequisite to lyophilization, the iron-delivery
vehicle or AHS present as an iron-saccharidic complex is preferably
separated from its coexisting hydrophilic and other excipients as
described previously. Such excipients include excess synthesis reactants,
reaction byproducts, waste glucans, polyglucans, saccharidic lactones,
degradation byproducts and other substances. In a preferred embodiment,
the AHS is separated, in the manner of the primary reference standard
species, comprising Fraction 1. By virtue of separating the AHS from
coexisting hydrophilic substances, there is an increase in the Aw
value of the fraction or composition in which it is present; in other
words, the Aw value approaches 1.0 in the AHS containing fraction.

[0077]Freeze drying technology is well known in the food processing
industry and has also been employed in the drying of pharmaceuticals. The
technology is typically applied in order to dry compositions that are
water-wet, although it is feasible to dry materials or solutes that are
dispersed or dissolved in other diluents or solvents, alone or in
admixture, for example, with water, and that are susceptible to freeze
drying. Generally, the composition is frozen to a temperature
significantly below 0° C. and subjected to a low absolute
pressure, in other words, a high vacuum. Heat is carefully introduced in
order to cause the ice to sublime. The process has been used to protect
heat sensitive materials from thermal damage as well as to prevent
shrinkage of porous materials during drying, so that they can be quickly
and fully rehydrated. The present invention provides a method for the
separation and lyophilization or freeze drying concentration of active
hematinic species manufactured for use as parenteral iron delivery
vehicles.

[0078]During freeze drying, a changing state of unbalance exists between
ice in the frozen composition, referred to as product ice, and system
pressure and temperature conditions. The migration of water vapor from
the product ice interface occurs only if this state of unbalance exists
and the product ice is at a higher energy level than the rest of the
system. Freeze drying equipment is designed to present a set of
controlled conditions effecting and maintaining the preferred temperature
and pressure differences for a given product, thereby causing the product
to be dried in the least amount of time.

[0079]The limit of unbalance is determined by the maximum amount of heat
which can be applied to the product without causing a change from solid
to liquid state (referred to as melt-back). This may occur even though
the chamber pressure is low since the product dries from the surface
closest to the area of lowest pressure; this surface is called the ice
interface. The arrangement of the drying, solid composition or particles
above this interface offers resistance to the vapors released from below,
thereby raising the product pressure and temperature. To avoid melt-back,
heat energy that is applied to the product closely approximates, and
preferably does not exceed, the rate at which water vapor leaves the
product. Another factor affecting the process is the rate at which heat
energy applied to the product ice (and carried away by the migrating
vapors) is removed by the condenser refrigeration system. By maintaining
a low condenser temperature, water vapor is trapped as ice particles and
effectively removed from the system, thereby reducing and simplifying the
vacuum pumping requirement. Air and other noncondensible molecules within
the chamber, as well as mechanical restrictions located between the
product ice and the condenser, offer additional resistance to the
movement of vapors migrating towards the condenser.

[0080]Four conditions are generally considered essential for freeze
drying. These process conditions are as follows: (1) the product is
solidly frozen below its eutectic point or glass transition temperature;
(2) a condensing surface capable of reaching temperatures approximately
20° C. colder than the ice interface temperature is provided,
typically less than about -40° C.; (3) the vacuum system is
capable of evacuation to an absolute pressure of from about 5 to about 65
microns of Hg (about 0.5 to about 10 Pa; preferably from about 1 to about
8 Pa); and, (4) a source of heat input to the product, controlled at from
about -60° C. to about +65° C.; preferably from about
-40° C. to about +65° C.; more preferably from about
-30° C. to about +55° C.; most preferably from about
-25° C. to about +25° C.; typically, a temperature of about
+20° C. is employed to provide the heat required to drive water
from the solid to the vapor state (i.e., the heat of sublimation). The
physical arrangement of equipment designed to satisfy these four
conditions varies widely, and includes individual flask freeze drying
apparatus and batch process freeze drying apparatus. Freeze drying
processes are typically carried out in chambers on a batch basis when
exacting control of the process is required, such as in the chemical and
pharmaceutical industry. This allows an operator to more precisely
control what occurs to the product being sublimed. Suitable equipment is
described, for example, in U.S. Pat. No. 6,122,836 (assigned to the
Virtis Division of S.P. Industries, Inc., N.Y.) and references cited
therein, as well as Zapsalis and Beck, Food Chemistry and Nutritional
Biochemistry, 1985, Chapter 1, pp. 23-26 (all of which are incorporated
herein by reference to the extent permitted). Other suitable commercial
equipment and process conditions are described in detail in the section
entitled "Freeze Drying", van Nostrand's Scientific Encyclopedia, Eighth
Edition, pages 1338-1342, 1995 (incorporated herein by reference to the
extent permitted).

[0081]The effectiveness of freeze drying processes is partially dictated
by the triple point curve for water where solid water in the form of ice
undergoes a direct transformation into the vapor phase at temperatures of
less than 0° C., and pressures of less than 4.58 Torr (610.5
Pascals). Efficient freeze drying is conducted under a pressure (vacuum)
of from about 10 microns to about 200 microns Hg; preferably from about
40 to 100 microns; more preferably from about 40 to about 80 microns;
typically a pressure of about 60 microns is used. The removal of water
molecules existing as (a) ice within a hydrated physical matrix or (b)
ice that develops from freezing simple aqueous solutions, ideally gives a
dry residual physical matrix free of water or a residue of some desired
water-free solute. However, where hydrophilic solutes, colloids,
suspensions or dispersions exist within an ice system, such as
saccharidic excipients, they can concentrate within the ice structure as
the ice is subjected to the freeze drying process and the volume of
solvent or diluent water is reduced. Since such materials become more
concentrated as the freeze drying progresses, this increasingly depresses
the freezing point of the frozen aqueous system. As this condition
proceeds, the colligative properties of solute interaction with water can
also rise above the eutectic point, contributing to or causing a
melt-back phenomenon. This is contrary to the preferred freeze drying
process of the present invention in which the ice accompanying the
desired solute, the AHS, is maintained in a frozen state, substantially
unimpaired by hydrophilic solute species that may include
difficult-to-remove water, until substantially all water associated with
the AHS has been removed by sublimation.

[0082]The preferred freeze drying of the AHS is accomplished, in
significant part, as a result of the high Aw of the fraction
(Fraction 1) comprising substantially all of the iron-saccharidic complex
originally present in the sample, which facilitates its rapid
shell-freezing onto a plate, the walls of a container or some other
three-dimensional scaffolding that ensures a high surface to volume ratio
for the frozen fraction. The more efficiently that shell-freezing occurs,
the better the quality of the lyophilized product. Freezing is typically
carried out at temperatures of from about -160° C. to about
-10° C.; preferably from about -80° C. to about -20°
C.; for example about -60° C. When the AHS is present in a frozen
composition where the water displays an Aw value approaching 1.0,
pressures below about 4.58 Torr (610.5 Pa) result in increased water
vapor pressure and temperature conditions as described can result in an
increased water vapor pressure and efficient sublimation. Water is
removed from ice by maintaining the pressure surrounding the frozen AHS
below the vapor pressure on the surface of remaining ice, removing the
water vapor with a vacuum pump and condensing it on refrigerated surfaces
held at temperatures of from about -120° C. to about -25°
C.; preferably from about -80° C. to about -50° C.;
typically -60° C. In particular, the high Aw of the
previously separated AHS facilitates the migration rate of the
sublimation front throughout the frozen product. In the absence of
removing the excipients, particularly hydrophilic excipients, from the
AHS or iron-saccharidic complex, the AHS is subject to melt-back during
the freeze drying process. In other words, the presence of hydrophilic
substances results in water being sufficiently bound or retained by the
AHS composition in which such hydrophilic substances are present. If
higher temperatures are employed to increase the vapor pressure in an
effort to remove such bound water, this also can have the undesirable
effect of causing the ice phase to melt, thereby impairing freeze drying.
Consequently, it is preferred that all or substantially all of the
hydrophilic excipients be removed or separated from the AHS prior to
freeze drying: preferably greater than about 95% of those originally
present are removed; more preferably greater than about 98%; still more
preferably greater than about 99%; most preferably greater than about
99.9% are removed; for example, the AHS is separated from hydrophilic
excipients prior to freeze drying to the extent that such excipients are
present in trace amounts.

[0083]For purposes of the present invention, the dried AHS residue
comprises the pharmacologically useful iron-saccharidic complex. Thus,
the separated and dried AHS is suitable for further analytical study or,
optionally, reconstitution, in order to meet other investigative
analytical or pharmacological uses. Typically, the methods of the present
invention are suitable for drying AHS, from which excipients have been
substantially removed, such that from about 85% to at least about 99%;
preferably from about 90% to at least about 97%; most preferably from
about 92% to at least about 95% of the water has been removed. It should
be appreciated that a small percentage of the water originally present in
the separated AHS may be associated, or strongly bound, to the AHS and
attempts to remove such bound water may pose a danger of unnecessarily
degrading the AHS. A sample of post-lyophilized and reconstituted AHS
subjected to LLS and RI-based HPLC analysis, is illustrated in FIG. 10.
The figure shows a chromatographic signature substantially identical to
that in FIG. 1, which serves as the primary reference standard. Moreover,
the analytes depicted in FIGS. 1 and 10 are essentially identical to the
AHS seen in FIG. 3 where excipients were allowed to remain.

[0084]A hematinic material with an HPLC profile that is different from the
primary reference standard such as in FIG. 1, or in FIG. 10, is one that
also shows evidence of the AHSAP in the lyophilized product having an
unusual morphology. When the AHSAP is observed to be present as part of
the AHS in the course of HPLC studies, the microscopic appearance of the
lyophilized AHS at 100-fold magnification and higher, can be visually
described as a corduroy-type structure. It displays red-brown parallel
bands or wales of ferric iron uniformly interspaced with transparent
bands of thin carbohydrate plates. The red-brown parallel bands of ferric
iron have a distinct columnar shape, parallel to each other, that are at
least twice the diameter of the thickness displayed by the long planar
transparent carbohydrate plates that are repeatedly interspaced between
them. This observed microscopic form has structural analytical
significance that corresponds to HPLC light scattering data when
chromatographic profiles appear as those seen in FIGS. 4-9. When a
desirable freshly prepared AHS corresponding to the primary reference
standard of iron-saccharidic complexes is present, the morphology of the
lyophilized product is characteristically different in that there is an
absence of columnar structure.

[0085]Preservation of the lyophilized product can be maintained in a
vacuum or under any inert gas, including, for example, nitrogen, argon
and helium (as well as any gas that is not reactive with the lyophilized
product) before it is reconstituted for analysis or use. Also, since the
lyophilization process alone does not compromise the structure of
iron-saccharidic complexes, use of the process has value for maintaining
these hematinic agents at various time intervals so as to document the
hematinic species present at a given point in time when lyophilization
was implemented. This provides a method for archival storage and
documenting of product manufacture and quality. In other cases,
lyophilization can be specifically used to stabilize and store primary
and/or secondary reference standards of these hematinic compositions.
Furthermore, suitably prepared and maintained lyophilized AHS can be
safely stored until needed with little risk of significant degradation of
the product. Furthermore, the product in such a form can be conveniently
shipped to geographically remote locations and conveniently stored until
needed, at which time reconstituting the hematinic for parenteral use is
readily accomplished. For example, the lyophilized product prepared
according to the present invention can be stored in sealed glass or
appropriately protected metal containers, preferably topped with a
substantially moisture free inert gas. Alternatively, such product can be
sealed in a metal foil pouch in a quantity suitable for reconstituting as
a single parenteral dose, etc. The iron-saccharidic complexes referred to
are prepared in order to produce parenteral hematinic complexes for the
delivery of iron to humans or animals in need thereof. These iron
complexes generally occur in a form such that iron can be parenterally
and benignly administered to augment hematopoietic mechanisms required
for the management of numerous clinical conditions in mammals,
particularly in human beings in need thereof.

[0086]Parenteral administration of a substance, e.g., a drug or the AHS of
the present invention, refers to introduction into the body by some means
other than through the gastrointestinal tract. In particular, it
includes, intravenous, subcutaneous, intramuscular or intramedullary
injection or short, e.g., about 5 minutes, or prolonged infusion, e.g.,
about 30 minutes or longer. Parenteral routes of administration can
provide benefits over oral delivery in particular situations. For
example, parenteral administration of a drug typically results in
attainment of a therapeutically effective blood serum concentration of
the drug in a shorter time than is achievable by oral administration.
This is especially true of intravenous injection, whereby the drug is
placed directly in the bloodstream. Parenteral administration also
results in more predictable blood serum concentrations of the drug,
because losses in the gastrointestinal tract due to metabolism, binding
to food and other causes are eliminated. For similar reasons, parenteral
administration often permits dose reduction. Parenteral administration is
generally the preferred method of drug delivery in emergency situations,
and is also useful in treating subjects who are uncooperative,
unconscious, or otherwise unable or unwilling to accept oral medication.
With regard to hematinics, parenteral administration of the AHS is
particularly useful for patients undergoing dialysis treatment since it
can be administered concurrently.

[0087]As described above, the separated AHS can be lyophilized and stored
as a freeze-dried composition. Thereafter an injectable solution can be
prepared by reconstitution of the composition. Furthermore an article of
manufacture can be produced comprising a sealed container such as a vial,
ampoule or pouch having contained therewithin a unit dosage amount of the
composition in a sterile condition. Such an article can be used for
treating or preventing an iron deficiency disorder in a subject, the
method comprising (a) reconstituting a unit dosage amount of the
composition in a physiologically acceptable volume of a parenterally
acceptable solvent liquid to form an injectable solution, and (b)
injecting the solution parenterally into the subject. At the time that
the AHS is reconstituted for parenteral use, additional agents or
excipients can be added in controlled amounts in order to provide a
suitable parenteral solution. Such agents include, for example, buffering
agents, pH modifiers, preservatives, tonicity adjusting agents, etc.
Alternatively, one or more of such agents can be included in an
appropriate amount in dry or powder form with the lyophilized AHS such
that when the AHS is reconstituted for parenteral use, the resulting
parenteral composition includes the necessary materials to form a
suitable parenteral composition for immediate use. Alternatively, only
the buffering agent is present and other agents are added to the extent
required.

[0088]One or more active hematinic species selected from those disclosed
hereinabove are present in a reconstitutable powder composition of the
invention in a total amount of about 30% to about 95%, alternatively
about 40% to about 90%, or about 50% to about 85%, by weight of the
composition.

[0089]When used, the buffering agent is present in an amount of about 5%
to about 60%, preferably about 10% to about 60%, and more preferably
about 20% to about 50%, by weight of the composition, and is typically
the predominant excipient ingredient. In one embodiment of the invention,
the reconstitutable powder composition consists essentially of the AHS
and the buffering agent.

[0090]The buffering agent is selected to provide a pH of the composition,
upon reconstitution in a physiologically acceptable volume of a
parenterally acceptable carrier or solvent liquid, that (a) is
parenterally acceptable, (b) is consistent with the AHS being in solution
or sufficiently dispersed so as not to cause an unacceptable adverse
reaction, in the carrier or solvent liquid, and (c) provides a medium
wherein the AHS exhibits acceptable chemical stability for at least about
one hour following reconstitution so as to facilitate parenteral
administration. Suitable buffering agents can illustratively be selected
from sodium and potassium phosphates, sodium and potassium citrates,
mono-, di- and triethanolamines,
2-amino-2-(hydroxymethyl)-1,3-propanediol (tromethamine), etc. and
mixtures thereof. Preferred buffering agents are dibasic sodium and
potassium phosphates and tromethamine. An especially preferred buffering
agent is dibasic sodium phosphate, for example dibasic sodium phosphate
anhydrous, heptahydrate, dodecahydrate, etc.

[0091]In one embodiment, the pH of the composition upon reconstitution is
about 7 to about 9, preferably about 7.5 to about 8.5, for example about
8. If desired, pH can be adjusted by including in the composition, in
addition to the buffering agent, a small amount of an acid, for example
phosphoric acid, and/or a base, for example sodium hydroxide.

[0092]Excipients other than the buffering agent, if present, constitute
not more than about 10%, preferably not more than about 5%, by weight of
the composition prior to reconstitution. For purposes of this discussion,
the term excipient does not include water. In one embodiment of the
invention, no excipients other than the buffering agent are substantially
present.

[0093]Optionally, one or more preservatives can be included in the
composition at up to about 0.5% by weight. Suitable illustrative
preservatives include methylparaben, propylparaben, phenol and benzyl
alcohol.

[0094]An injectable solution composition prepared by reconstituting a
powder composition as herein provided in a parenterally acceptable liquid
carrier or solvent, preferably an aqueous solvent, is a further
embodiment of the present invention. Any known parenterally acceptable
liquid carrier or solvent can be used to reconstitute a powder
composition of the invention. Water for injection can be suitable, but
will generally provide a hypotonic solution. Accordingly, it is generally
preferred to use an aqueous liquid containing a solute such as sodium
chloride and/or possibly dextrose. Illustratively, 0.9% sodium chloride
injection USP, sterile 0.9% sodium chloride injection USP, 5% dextrose
injection USP, and 5% dextrose and 0.45% sodium chloride injection USP
are suitable.

[0095]A suitable volume of the liquid carrier or solvent for
reconstitution depends on the age and body weight of the subject, the
solubility and dosage amount of the AHS and other factors, such as
whether the parenteral composition is to be administered by injection, IV
push or IV.

[0096]In this process, AHS and dibasic sodium phosphate heptahydrate as
buffering agent are dissolved in water to form an aqueous solution or
composition. Preferably water for injection is used as the solvent. AHS
and the buffering agent are present in the solution at concentrations
relative to each other consistent with the desired relative
concentrations of these ingredients in the final composition. Absolute
concentrations of these ingredients are not critical; however, in the
interest of process efficiency it is generally preferred that the
concentration of AHS be as high as can be conveniently prepared without
risking exceeding the limit of solubility to the extent of forming an
unsuitable aggregate. Other parenteral formulation ingredients or agents
as described above can be added in this step if desired. Order of
addition is not critical

[0097]An article of manufacture comprising a sealed vial, preferably a
glass vial, having enclosed therewithin a powder composition as herein
provided in a unit dosage amount and in a sterile condition, is a further
embodiment of the present invention. In a particular embodiment, such an
article of manufacture is provided, prepared by a process as described
above. The vial preferably has a capacity sufficient to enable
reconstitution of the composition in situ. Generally a capacity of about
1 ml to about 10 ml, preferably about 2 ml to about 5 ml, will be found
convenient. The term "vial" herein is used to denote any small container,
having a closure, that is suitable for packaging a unit dosage amount of
a reconstitutable powder, preferably in a sterile condition. It will be
understood that equivalent forms of packaging, such as an ampoule, a
disposable syringe and a syringe cartridge, are encompassed by this
embodiment of the invention.

[0098]The present invention is further directed to a therapeutic method of
treating a condition or disorder where treatment with a hematinic is
indicated, the method comprising parenteral administration of a
reconstituted composition of the invention to a subject in need thereof.
The dosage regimen to prevent, give relief from, or ameliorate the
condition or disorder preferably corresponds to any suitable interval in
accordance with a variety of known factors. These include the type, age,
weight, sex, diet and medical condition of the subject and the nature and
severity of the disorder. Thus, the dosage regimen actually employed can
be varied.

[0099]A typical preparation comprising an AHS prepared according to the
process of the present invention and provided in a suitable container,
e.g., an ampoule, vial or pouch, generally contains sufficient AHS so as
to provide, upon reconstitution, about 5 to 100, e.g., about 7 to about
50, typically about 10 to about 40 mg iron per mL.

[0100]A parenteral AHS in the form of sodium ferric gluconate can be
produced in a composition equivalent to that of a presently available
commercial product, for example, in the presence of sucrose.
Consequently, the composition can be administered in a dosage form and
based on an administration schedule equivalent to that currently
recommended. The dosage is typically expressed in terms of the mg content
of elemental iron. For example, the recommended dosage for repletion of
iron deficiency in hemodialysis patients is equivalent to 125 mg of iron
for a single administration. The product, when provided in the form of a
5 mL ampoule for intravenous injection containing 62.5 mg (12.5 mg/mL) of
elemental iron and also containing approximately 20% sucrose w/v (195
mg/mL) in water at a pH of 7.7-9.7, can be administered as a 10 mL dose;
equivalent to 125 mg of elemental iron. For slow IV administration
(undiluted), 125 mg can be introduced over 10 minutes; for IV infusion
(diluted in 0.9% NaCl), 125 mg in 100 mL over 60 minutes. A physician
trained in the art can determine the appropriate total dosage needed by a
patient based on the medical and physical condition of the patient and
the iron improvement required. For example, in order to achieve a
favorable hemoglobin or hematocrit response, the current recommendation
for the commercial hematinic of the above type is a minimum cumulative
dose of 1.0 gram of elemental iron, administered over eight sessions at,
e.g., eight sequential dialysis treatment sessions.

[0101]Dosage and administration of a parenteral product based on another
currently available commercial product in the form of sodium ferric
hydroxide in sucrose is also described in the art. Dosage of this form is
also typically expressed in terms of elemental iron content. Typically
each 5 mL vial of the composition contains 100 mg of elemental iron based
on 20 mg/mL. Repletion treatment of iron deficiency in hemodialysis
patients is typically 5 mL comprising 100 mg of elemental iron delivered
intravenously concurrent with dialysis. Patients typically require a
total of 1 gram (1,000 mg) of elemental iron administered in conjunction
with 10 sequential dialysis sessions for an appropriate hemoglobin or
hematocrit response. Maintenance of appropriate levels of hemoglobin,
hematocrit and other laboratory criteria may be determined by a skilled
physician, as appropriate.

[0102]The term "about" when used as a modifier for, or in conjunction
with, a variable is intended to convey that the numbers and ranges
disclosed herein are flexible and that practice of the present invention
by those skilled in the art using temperatures, concentrations, amounts,
contents, carbon numbers, properties such as molecular weight, viscosity,
solubility, etc., that are outside of the range or different from a
single value will achieve the desired result, namely preparation of a
hematinic iron-saccharidic complex suitable for freeze drying, the highly
purified hematinic iron-saccharidic complex produced thereby and methods
for its use. Furthermore, where a range of values is expressed, it is to
be understood, unless otherwise expressed, that the present invention
contemplates the use of the other ranges that are subsumed within the
broadest range.

Examples

[0103]For purposes of the present invention, reference to water content of
an undried substance or composition, in other words, prior to being
dried, is given as a percentage of the total weight of the undried
substance or composition. Water content of a dried substance or
composition is given as a percentage of the total weight of dry matter
only, excluding all moisture.

[0104]Following is the procedure for low pressure gel permeation
chromatography used in preparing the samples for which test results
appear in FIGS. 1, 2 and 10, including preparation of purified,
substantially excipient-free AHS. The specific application of low
pressure gel permeation chromatography (GPC) for AHS separation employs
crosslinked polyglucans or dextrans displaying molecular weight exclusion
characteristics greater than about 5,000 and preferably greater than
about 1,500 Daltons. The stationary GPC phase is "Sephadex G-10"
(Amersham-Pharmacia Biotech, Piscataway, N.J.). A solvent reservoir
supplies a mobile phase of HPLC grade water by gravity or metered flow to
a GPC column containing the stationary dextran phase. The column is
constructed of glass having a 2.0 cm diameter and a length of 25 cm. The
stationary phase is prepared according to manufacturer recommendations
including hydration of the dextran and vacuum degassing before use. A 400
microliter sample volume of the hematinic solution, including the
iron-saccharidic complex, for example, as released by its manufacturer in
sealed glass ampoules, is fed to the top of the GPC column and allowed to
permeate into the stationary phase. Once the highly colored hematinic
solution has penetrated into the stationary phase, HPLC grade water is
supplied manually or by pump at 1-4 mL per minute so as to ensure its
elution as a well-defined color band through the column. When the
characteristically colored AHS has eluted from the column as determined
by minimal spectrophotometric absorption at 430 nm this marks the end of
elution for Fraction 1. A more refined analytical method for finding a
separation point for Fraction 1 and the beginning of the subsequent
fraction identified herein as Fraction 2 uses the anthrone reaction
(Dreywood, 1946 cited previously). The eluate boundary between the two
Fractions can be defined because the lowest concentration of
furfural-producing carbohydrates in the overall eluate flow of the
partitioning process occurs between the AHS and its hydroscopic
excipients. In practice, 100 microliter samples of the eluate flow are
sampled, reacted with anthrone reagent and the resulting 620 nm
absorbance is recorded. The red-brown colored Fraction 1 contains the AHS
substantially free of hydrophilic and highly hydroscopic excipients that
formerly coexisted with the AHS as released. Remaining volume eluted from
the column is regarded as Fraction 2.

[0105]The AHS obtained, for example, from Fraction 1 as described above,
or from samples taken directly from glass ampoules of hematinic
compositions distributed for use in clinical applications or samples
prepared from concentrated volumes of Fraction 1, including reconstituted
compositions based on the use of freeze drying, can be further analyzed
with the use of HPLC-based refractive index (RI) and laser light
scattering (LLS) analysis. Specifically, the method uses a Waters 590
pump (Waters Corporation, Milford Mass.) to supply an aqueous mobile
phase to a 7.8 mm diameter by 30 cm long GMPWXL column (Tosoh
Biosep, Montgomeryville, Pa.). The internal column support material is
comprised of polymethylmethacrylate backbone eliminate polymer beads
having a 13 micron diameter particle size with a various pore sizes in
the range of from less than about 100 Angstroms to about 2000 angstroms.
The column eluant stream is monitored by a Wyatt miniDawn multi-angle
light scattering detector in combination with an Optilab DSP
interferometric refractometer (both from Wyatt Technology, Inc., Santa
Barbara, Calif.). The column heater and refractometer operating
temperatures were held at 35° C. The aqueous mobile phase included
200 parts per million sodium azide, pH was adjusted to 6.0 and was
subjected to 0.02 micron vacuum filtration and an ebullient helium sparge
before being used. The mobile aqueous phase was supplied to the system at
a flow rate of 1.0 mL per minute with a pressure of 150 pounds per square
inch. Preparation of a sample for testing requires 0.02 micron filtration
through a membrane filter (for example, "Anotop" filters, Whatman,
Maidstone, England). As hematinic compositions, iron-saccharidic
complexes or components thereof age, membrane filters up to 0.45 microns
may be required in order to remove larger particulates without clogging.
If particulates are not eliminated from analytical samples before
injection into the HPLC system, HPLC analytical performance will be
severely corrupted. Samples are diluted as desired up to 2.5% weight by
weight and a 80 to 200 microliter sample volume is injected into the HPLC
system for analysis. For multiple sample analyses, automation is
facilitated by use of "Water's autosampler", model 717 (Milford, Mass.).

[0106]The combination of RI and LLS detection with HPLC establishes an
absolute macromolecular weight for analytes that produce a
chromatographic peak as well as a root mean square (rms) radius value
also referred to as a radius of gyration (Rg). The rms value coupled with
absolute weight determination provides insight into the shapes of light
scattering species, such as rods, coils, spheres or discs. The formula
weight of the AHS and shape of specific iron-saccharidic complexes can be
used for various monitoring purposes.

[0107]The freeze drying process used for the examples of the present
invention is as follows:

[0108]Fraction 1, identified above, serves as the starting material for
freeze drying. Using the method described below, volumes as small as 10
mL or as large as 100 mL and comprising the AHS can be readily freeze
dried provided that the sample is substantially free of saccharidic
substances that tend to decrease the entropy of water and its vapor
pressure. These volumes can be contained in any glass container that will
withstand the physical stress of shell freezing, which method is used to
expedite the overall dehydration and concentration of the AHS. The
preferred ratio of liquid volume to the container volume for shell
freezing is from about 1 to about 5, but other ratios are feasible. After
liquid containing the substantially excipient-free AHS is introduced into
the container, the container is rotated at about 50 revolutions per
minute in a cryogenic bath. The bath can be made by mixing dry ice and
acetone or, alternatively, liquid nitrogen can be used, provided that a
temperature of at least about -50° C. or lower is maintained. The
immersion and rotation of the container freezes the AHS-containing
aqueous volume onto the walls of the container. This increases the
surface to volume ratio of the AHS-containing aqueous volume so as to
expedite water sublimation. Other process and equipment variations of
this procedure can be used to obtain the same or similar results.

[0109]One or more containers of shell-frozen compositions comprising water
and AHS are situated on a shelf within a freeze dryer. An instrument such
as a "Virtis Unitop 600L" linked to a "Freezemobile 12 ES" (Gardiner,
N.Y.) can be used for this purpose. A vacuum of 60 microns of Hg (7.5 Pa)
was maintained in the system and a condenser temperature of at least
about -60° C. or colder was maintained. A preferred freeze drying
cycle was as follows: initial shelf holding temperature of -50° C.
for 2 hours; temperature ramped up to 25° C. over a 12 hours;
sample soak at 25° C. for an additional 24 hours. Preferably the
dried product should be stored under desiccating storage conditions, for
example under a dry, inert gas such as argon or nitrogen. The dried AHS
can be reconstituted with a desired aqueous volume whereupon it readily
goes into a solution and can be readily filtered through a 0.02 micron
membrane, as described above.

[0110]HPLC analysis using RI and LLS detection is demonstrated in the
following 10 examples using iron-saccharidic complexes. The results for
each of samples 1 through 10 corresponds to FIGS. 1 through 10. The
hematinic samples 1-4 and 6-10 are the iron-saccharidic complex
identified as sodium ferric gluconate complex in sucrose (SFGCS), sold
under the brand name Ferrlecit® (manufactured by Rhone-Poulenc Rorer,
Dagenham, Essex, England). Sample 5, and its corresponding FIG. 5, is
ferric hydroxide sucrose complex (FHSC), sold under the brand name
Venofer® (manufactured by Luitpold Pharmaceuticals, Shirley, N.Y.).
Samples used for HPLC analysis were taken from newly opened glass
ampoules stored at room temperature conditions. Additionally, samples 6,
7, 8, and 9 were analyzed after 6, 12, 22 and 25 months following
manufacturing release of the product. These time periods are referred to
herein as "time after release", TAM and correspond to TAM.sub.#1,
TAM.sub.#2, TAM.sub.#3 and TAM.sub.#4 in the examples. Samples were
prepared by a 1 to 20 dilution and 200 microliters sample volumes of
these dilutions were analyzed by the HPLC method specified above.

[0111]Example 1 test results are shown in FIG. 1. The results are based on
the use of HPLC with RI and LLS detection for evaluating an AHS isolated
in the Fraction 1 eluate obtained using preparative low pressure GPC and
a sample of iron-saccharidic complex as obtained from its glass
distribution ampoule. The single well defined chromatographic profile for
the LLS signal and the RI signal coincide for AHS elution but no other
excipients appear in the purified material.

[0112]Example 2 results are shown in FIG. 2. The results are based on the
use of HPLC with RI and LLS detection for evaluating the AHS isolated in
the Fraction 2 eluate obtained from using preparative low pressure GPC
and the iron-saccharidic complex of Example 1. It is clear from these two
results that the excipients and AHS are separated or isolated in distinct
fractions. 15 microliters of AHS from Fraction 1, Example 1 was added to
the Fraction 2 eluant as an internal standard in order to identify where
its elution position would appear relative to that of the excipients.

[0113]Example 3 results are shown in FIG. 3. The results are based on the
use of HPLC with RI and LLS detection, applied to the same hematinic
sample as in Example 1, to substantially separate Fraction 1 with its
characteristic AHS, from excipients usually observed in Fraction 2. The
HPLC method can discern the various iron-saccharidic complex constituents
of a hematinic composition on a single chromatographic profile. While
HPLC is particularly suited to rapid analytical testing, low pressure
chromatography is particularly suited as a preparative method for the
preferred AHS. The LLS signal for the AHS corresponds to the observed RI
signal.

[0114]Example 4 results are shown in FIG. 4. The results are based on the
use of the preferred HPLC method for detecting structural deviations from
the original AHS. In its undegraded form, the AHS serves as a quality
benchmark also denoted in these Figures as a "primary reference
standard". HPLC analysis using RI and LLS was carried out on a hematinic
composition obtained directly from its delivery ampoule and comprising an
iron-saccharidic complex. The figure shows inconsistencies in the
expected, or ideal, AHS peak. Note the appearance of a new, observable
chromatographic secondary peak adjoining that of the AHS primary
reference standard. This feature is indicative of iron aggregate species
as a consequence of AHS degradation. The figure identifies the second
peak as an active hematinic species aggregate peak (AHSAP) using LLS
detection. It is particularly noteworthy that this peak is not observed
using RI detection alone.

[0115]Example 5 results are shown in FIG. 5. The results are based on the
use of HPLC equipped with RI and LLS detectors. This figure also shows
structural changes in the AHS, or primary reference standard peak, of
hematinic comprising FHSC. The sample of this product included a
manufacturing date of December 1999 on the ampoule and an expiration date
of December 2002. In this example, the AHSAP appears as a shoulder on the
AHS peak, suggesting a different degree of change compared with the
sample studied in Example 4. While LLS and RI chromatographic profiles
generally overlap, only the LLS signal detects evidence of iron
aggregates in the parenteral hematinic. As in each previous example, the
sample for this study was obtained directly from a sealed glass ampoule
used for clinical distribution.

[0116]Examples 6-9. Readily detectable departures in the HPLC based RI and
LLS elution profiles from the expected chromatographic profile for an AHS
reflect either a departure from preferred manufacturing conditions or
degradation of AHS due to aging and destabilization while still sealed in
glass delivery ampoules. The destabilization reflects itself in the
aggregation of iron normally embodied as a constituent of the desirable
AHS structure. Although HPLC with RI detection fails to detect the
changes in the iron-saccharidic complexes, LLS detection clearly provides
evidence of such product destabilization. The advantages of the invention
as a method for monitoring the state of a hematinic product comprising
iron-saccharidic complex is illustrated where HPLC is coupled with RI and
LLS detectors in order to detect evidence of degradation of the AHS as
indicated by iron aggregate formation. Iron aggregate formation is
observed as the HPLC chromatographic peak denoted as AHSAP. Individual
samples of iron-saccharidic complexes, specifically SFGCS, manufactured
over the course of several months were stored at room temperature in the
absence of light and without any excursions known to stress the stability
of the products while sealed in their glass ampoules. The samples were
aged at room temperature in the dark and after 6, 12, 22 and 25 months
following their manufacture, the respective ampoules were opened and the
contents analyzed by HPLC with RI and LLS, as described. None of the
stored product samples had reached its stated expiration date stated on
the packaging material. The sample with the shortest TAM value of 6.0
months was designated as TAM.sub.#1 and that with the longest storage of
25 months TAM.sub.#4. The results of HPLC studies applied to this range
of successively aged hematinic examples are shown in FIGS. 6-9. The key
area of analytical interest in the chromatographic profiles presented in
FIGS. 6-9 is the region where the AHS signature appears, thus only that
specific elution range pertinent to the RI and LLS analytical profile is
shown.

[0117]Taken as a group in sequence from TAM#1 (FIG. 6) through TAM#4 (FIG.
9), it is apparent that the RI signal from these samples show little
effect of sample aging by way of AHS decomposition and iron aggregation.
On the other hand, evidence of AHS destabilization with iron aggregation
is prominently seen by the AHSAP shoulder or a secondary peak, which was
detected by using LLS in all four samples.

[0118]By way of these examples, it is evident that the preferred HPLC
based RI and LLS method provides an ability to verify the presence of an
AHS or its coexistence with normally occurring excipients with which it
is released for parenteral use. Beyond this, the method affords a
significant ability to monitor, as well as to investigate and develop,
hematinics based on iron-saccharidic complexes as a group. It can be seen
that this class of hematinics is susceptible to destabilization resulting
in iron aggregates that are commingled with the preferred or normal AHS.
Unless HPLC is used with at least LLS detection as well as RI detection,
the occurrence of iron aggregates can go unnoticed in these hematinic
agents.

[0119]As described above, when carrying out the preferred method of this
invention, samples are routinely filtered through a 0.02 micron Anotop
brand membrane filter to avoid operational problems with the sample
before it is injected into the HPLC. Iron-saccharidic samples that show
no evidence of AHSAP occurrence can be readily filtered in preparation
for study but older samples filter with great difficulty or not at all
even using 0.45 micron filters. It has been observed that difficulties in
the preparatory filtration of samples for HPLC study according to the
specified and preferred method correspond with the occurrence of the
highest levels of AHSAP. The measurable and quantitative entrainment of
iron aggregates over a membrane filter surface can provide another,
albeit cursory, method for evaluating the unintended degree of hematinic
breakdown. However, application of such a filtration method to a
hematinic before parenteral use would be impractical and, furthermore, it
would not have any effect on degraded AHS that had not progressed to the
filterable iron aggregate stage. When significant amounts of iron
aggregates develop in samples and hematinic analysis is essential, sample
filtration is necessary for HPLC instrument performance and maintenance.
Also, it should be noted that if a residue of iron aggregate or other
particulate material is entrained on a membrane filter as a result of
preparing AHS for HPLC analysis, the quantitative amount of aggregate on
the filter should be considered together with HPLC analysis as
complementary indicators of product decay. Where little or no evidence of
filterable material is present in hematinics and particulates have
dimensions of less than 10 nm in diameter, the HPLC method more
accurately serves as the preferred method for monitoring hematinic
quality.

[0120]Example 10 results are shown in FIG. 10. This example involved the
application of the preferred method for HPLC based RI and LLS analysis to
an AHS that was reconstituted from a freeze dried state. While AHS
isolated from an iron-saccharidic complex has never before been reported,
its ability to be freeze dried and reconstituted without decomposition,
degradation or iron aggregate formation was particularly uncertain. An
original 2.5 mL sample volume of iron-saccharidic complex as released,
SFGCS, taken from a sealed glass ampoule was separated into Fraction 1
and Fraction 2 according to the low pressure chromatography method for
preparing substantially excipient free AHS. Fraction 1 containing the AHS
was freeze dried as described above. One week after freeze drying it was
reconstituted to its original volume (2.5 mL) with HPLC grade water. A
500 microliter volume of the 2.5 mL reconstituted solution was then
diluted to 20.0 mL and 200 microliters of this was injected for HPLC
based RI and LLS analysis as described. The resulting chromatographic
profile is shown in FIG. 10. It is noted that both the LLS and RI signals
not only overlap, consistent with the chromatographic profiles observed
in FIG. 1, but there is no evidence of any iron aggregate formation or
AHSAP as observed in FIGS. 6-9. By way of this example, HPLC with RI and
LLS detection is also shown to be useful for monitoring the quality of
freeze dried AHS.

[0121]Example 11 was carried out to compare the response to freeze drying
of untreated versus treated hematinic. Three samples of an
iron-saccharidic complex, SFGCS, as manufactured and comprising AHS,
sucrose and residual excipients were subjected to freeze drying. All
three samples originated from the same production batch as released in
different glass ampoules. The freeze drying method used was the same as
that applied to the hematinic in Example 10, but contrary to that
example, the AHS was allowed to remain with its excipients during the
course of freeze drying. The experimental objective was to observe
whether or not there were any weight disparities in the final freeze
dried product due to the presence of hydrophilic excipients compared to
the same product freeze dried in the absence of such hydrophilic
excipients. Results are summarized for the hematinic dried with its
excipients in Table A.

[0122]Three additional samples of the same hematinic batch from the same
source as used in Table A were obtained from different unopened ampoules
and subjected to treatment in a low pressure chromatographic column as
described above. Fraction 1 of each sample (comprising the AHS, but
without the associated hydrophilic excipients, including sucrose) was
then subjected to freeze drying as described above; results are
summarized in Table B:

[0123]The test results clearly show that a significantly higher percentage
of water is removed from the samples subjected to column separation and
identical conditions of freeze drying.

[0124]Although the invention herein has been described with reference to
particular embodiments, it is to be understood that these embodiments are
merely illustrative of the principles and applications of the present
invention. It is therefore to be understood that numerous modifications
may be made to the illustrative embodiments and that other arrangements
may be devised without departing from the spirit and scope of the present
invention as defined by the appended claims.